Báo cáo khoa học: Transport Machineries docx

Transport Machineries B1-1 Osteopontin expression in polarized MDCK cells N. Tascene 1 and Z. S. Isguder 2 1 Ankara University Faculty of Veterinary Medicine, Ankara, TURKEY, 2 University of Veterinary Medicine, Department of Physiological Chemistry, Hannover, GERMANY Osteopontin(OPN) is an arginine-glycine-aspartate (RGD)-contain- ing adhesive glycoprotein that was first identified as a major sialo- protein in bone and subsequently found to be expressed in kidney, brain, macrophages, vascular smooth muscle cells and many cells of epithelial linings. In this study we examined the expression of OPN in MDCK cells in different cellular confluences. The cells were examined at very low density, half confluence or complete confluence. OPN levels were investigated by western blotting and RT-PCR. An increase in OPN expression was observed due to the increasing confluency and subsequent initiation of polarization. Expression profiles of flotillin-2 in the same cells was used as a control, since this protein is ubiquitously produced in MDCK cells and its expression rates are independent of confluence and/or polarization. Intracellular distribution of OPN was also monitored by confocal microscopy on preparations immunolabeled with anti- OPN antibodies. Staining patterns have also confirmed increased OPN expression during confluency in MDCK cells. We conclude that the expression of OPN in polarized MDCK cells is induced by increasing confluency and polarization. Key words: Osteopontin, polarization, MDCK cells. B1-2 Biogenesis of VGF se cretory granules in epithelial thyroid cells F. Gentile 1 , A. Corteggio 2 , S. Avitabile 2 , G. Calı ` 1 and L. Nitsch 2 1 IEOS, CNR, Napoli, ITALY, 2 University Federico II, Napoli, ITALY VGF is a granin that is sorted to a regulated and polarized path- way of secretion when expressed in FRT epithelial cells. We are investigating the role of aggregation and interaction with lipid rafts in VGF protein sorting. We demonstrated that a fraction of the intracellular protein is associated to GM1 rafts and that in a low pH and high calcium buffer VGF aggregates. Since only the intra- cellular protein, not the one secreted in the culture medium, is cap- able to aggregate, we hypothesized that VGF aggregation might be due to its interaction with some other molecule. We have excluded that the interaction of VGF to GM1 rafts is relevant for VGF aggregation since it is not affected by the inhibition of glyco- sphyngolipid synthesis. We have then looked for proteins capable to interact with VGF by gel filtration and by SDS-PAGE analysis of proteins that co-aggregated with VGF. We found a 40 kDa pro- tein that is co-eluted with VGF and we demonstrated that it is not flotillin 2, which also forms aggregates but has an intracellular dis- tribution distinct from that of VGF. FRT-VGF cells were treated with bafilomycin A1 that alters the proton gradient of the secre- tory compartment. A dramatic reduction in VGF secretion and granule formation, and lack of response to PMA were observed. The secretion of thyroglobulin, that we have stably expressed in FRT cells and that is also secreted through the apical cell domain but by a constitutive pathway, was unaffected. Overall our data support the hypothesis that VGF aggregation does not depend on rafts association and possibly occurs in the presence of a 40 kDa protein. Proton gradient perturbation affects granule formation and VGF secretion. B1-3 Cellular trafficking of cell penetrating peptides and protein transport over plasma membrane A. Lorents 1 , K. Padari 2 , M. Hansen 3 ,U ¨ . Langel 3 and M. Pooga 4 1 Institute of Molecular and Cell Biology, University of Tartu, Tartu, ESTONIA, 2 Institute of Zoology and Hydrobiology, University of Tartu, Tartu, ESTONIA, 3 Institute of Neurochemistry, Stockholm University, Stockholm, SWEDEN, 4 Estonian Biocentre, Tartu, ESTONIA Cell penetrating peptides (CPP) consisting of up to 30 amino acids are capable of translocation into cells and delivering different car- goes. Despite numerous efforts the internalization mechanism of CPPs has not been fully understood. The cellular uptake of CPPs is dependent on the properties and concentration of peptide, cellular energy and lipid-rafts of plasma membrane. Binding to the cell sur- face proteoglycans, followed by endocytic uptake is considered as the main mechanism of highly cationic CPPs’ cell-entry. However, the type of endocytosis induced by CPPs, further intracellular target- ing, and liberation from vesicular structures are still under debate. We assessed the protein delivery by CPPs in suspension cells K562 by flow cytometry and fluorescence microscopy. Among the studied peptides Tat and transportan showed highest protein transduction efficiency. The CPP-protein complexes localized in K562 cells both in vesicles and diffusely in the cytoplasm as demonstrated by fluores- cence microscopy. The inhibitors of macropinocytosis and cellular metabolism decreased the uptake of complexes by K562 cells in a concentration dependent manner, indicating that CPP-avidin con- structs are taken up mostly by energy-dependent mechanisms, invol- ving different types of endocytosis. The cellular trafficking of CPP-s in HeLa cells was mapped on ultra-structural level by transmission electron microscopy by using nanogold-labelled peptides. B1-4 Cellular uptake and subcellular distribution of Ap n A family of signaling molecules R. Pe˛cherzewska, A. D. Krakowiak, J. Kaz´ mierczak, M. Maszewska and W. J. Stec CMMS PAS, Lodz, POLAND Diadenosine polyphosphates are recognized as intra- and extracel- lular signaling molecules and considered as the class of new second messengers. Biochemical function of dinucleoside tri- and tetra- phosphates has not been fully elucidated yet [1]. It is known that Ap 3 A is cleaved by specific enzymes of hydrolases family. One of them is the tumor suppressor Fhit protein. It is assumed that for- mation of the FhitAp 3 A complex, recognized as a signaling mole- cule induces cell apoptosis [2]. The aim of presented studies is to determine the cellular uptake of the Fhit protein substrates. For our experiments we used fluorescent ana- logs of Ap n A. Thus, ApppBODIPY and GpppBODIPY containing the fluorophore residue (BODIPY) instead of one adenosine moiety as well as an analog of inhibitor of Fhit protein [3] conjugated with Alexa Fluor 350 have been used. Transfections were done with Lipo- fectamine 2000. Intracellular distribution of fluorescent analogs of diadenosine triphosphate was visualized by fluorescence microscopy. It has been found that both substrates with fluorescent residue, ApppBODIPY and GpppBODIPY, accumulate in cytoplasmic compartments and in the nucleoli of the transfected cells. Detailed results will be presented. Acknowledgement: This project is financially assisted by the grant 2PO4A07929 (to A.K.). References 1. Kisselev L. et al., FEBS Lett. 427: 157–63 (1998). 2. Brenner C. Biochemistry 41: 9003–9014 (2002). 3. Varnum J.M. et al., BMC Chem Biol, 1(1):3, (2001). Transport Machineries Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 97 B1-5 Characterization of heparan sulfate as a cell surface attachment molecule for human eosinophil ribonucleases S. C. Lin, T. C. Fan and M. D. T. Chang Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu, TAIWAN The eosinophil appears to be the primary leukocyte responsible for tissue damage in bronchial asthma, which occurs when the granule proteins, including eosinophil cationic protein (ECP) and eosino- phil derived neurotoxin (EDN), are released into the extracellular space. ECP and EDN belong to the ribonuclease A superfamily. Both have been suggested as factors in allergic respiratory diseases, and thus used as clinical biomarkers for detecting the severity of asthma. Interestingly, ECP and EDN showed cytotoxicity toward several cell lines, including HL60 and A431 cells. The cytotoxicity was positively correlated with the internalization properties of the RNases, but the exact route by which eosinophil RNases enter cells has not been clarified. Recently, we hypothesized that ECP and EDN may internalize target cells through binding to the cell surface heparan sulfate (HS). In this study, we have investigated the interaction between ECP/EDN and HS using surface plasmon resonance instrument (SPR) technology. On the other hand, amino acid sequence analysis reveals that EDN contains one putative heparin-binding motif of the type XBBXBX, whose effects on HS binding has been investigated by site-directed mutagenesis and binding assays. Our results indicate that the heparin-binding region plays a critical role in recognition and internalization of eosinophil RNases. B1-6 A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein T. Fan, S. Lin and M. D. T. Chang National Tsing Hua University, Hsin-chu, TAIWAN Eosinophils secrete several basic proteins, among which the eosino- phil cationic protein (ECP) has been used as bio-markers for the severity of asthma. ECP, which belongs to the human RNaseA su- perfamily, is released from activated eosinophils during the inflam- mation process and contributes to eosinophils’ helminthotoxic, bactericidal and antiviral activities. The cytotoxicity of ECP is clo- sely associated with its efficient endocytosis into target cells. We have previously discovered that CPE functions as a cellular receptor for ECP binding to cells. The exact endocytic pathway taken by ECP has not yet been deciphered. In particular, whether proteins of the RNase family are internalized by a raft-associated pathway is unknown. In this study, some key elements regulating the early events of internalization of ECP were discovered by characteriza- tion of binding, uptake and intracellular trafficking of ECP in human bronchial epithelial cells. Uptake and cytotoxicity of ECP in glycosaminoglycan-deficient cells were significantly reduced. After associating with cell surface heparan sulfate, ECP was found to rapidly internalize through detergent-resistant lipid rafts in a clathrin- and caveolin-independent fashion, followed by trafficking from early endosomes to late endosomes. B1-7 Activation of the ubiquitin ligase Itch following treatment with the epidermal growth factor B. A. Azakir and A. Angers University of Montreal, Montreal, PQ, CANADA Itch is an ubiquitin ligase localized at the endosomes and implica- ted in the regulation of endocytosis. Many substrates of Itch have been identified, including the endocytic proteins endophilin and cbl, p73, Smad-7 and others. Here we show that stimulation with EGF increases cbl, endophilin and Smad-7 ubiquitination mediated by Itch and that this ubiquitination is sufficient for the degradation of these proteins in the proteasomes. Moreover, Itch itself is regu- lated by EGF treatment, Itch ubiquitination increasing transiently after treatment. However, Itch is not degraded in the proteasomes, but deubiquitinated by the ubiquitin protease FAM. It has recently been reported that Itch can be phosphorylated by the c-Jun N-ter- minal kinase (JNK), and that this resulted in a significant increase in the ligase activity. It is known that JNK can also be activated by EGF treatment. Likely, we have found that Itch phosphoryla- tion is increased after treatment with EGF and that inhibition of JNK blocks this effect. Remarkably, Itch substrate ubiquitination after EGF treatment is also reduced after JNK inhibition. As Itch phosphorylation influences its capacity to interact with its sub- strates, we examined the interactions of Itch with endophilin, cbl, and FAM after EGF treatment. We show that Itch interaction with Cbl and endophilin increases after treatment with EGF, but interaction with FAM decreases. These results explain the transient ubiquitination of Itch and the sustained ubiquitination of its sub- strates after treatment with EGF. We conclude that Itch activity and stability is regulated by EGFR via JNK and that this regula- tion affects the ubiquitination and the degradation of Itch sub- strates, especially cbl and endophilin, which could in turn regulate receptor internalisation. B1-8 Identification of a novel Musk binding protein with a potential role in intracellular trafficking processes B. Woller and R. Herbst Medical University Vienna, Vienna, AUSTRIA The muscle-specific kinase MuSK plays an essential role during the formation of the neuromuscular junction. MuSK is activated by the nerve-derived protein agrin, a process that is essential for all known aspects of postsynaptic and presynaptic differentiation. The steps that follow MuSK activation and lead to acetylcholine recep- tor clustering are not fully understood. Therefore we have a high interest in the identification and characterization of new MuSK binding partners. Using a mouse muscle cDNA library we performed a yeast-two- hybrid screen with the cytoplasmic region of MuSK. Thereby we have identified a protein that specifically interacts with MuSK and represents a novel gene, termed Cl-II6. Current data suggest that the newly identified protein is a new member of the RIN protein family, that functions as GEFs for Rab5. We therefore propose that the novel protein is able to link MuSK to the endocytic machinery via Rab5. When a GFP-tagged form of Cl-II6 is expressed in heterologous cells, a punctuated intracellular staining is observed that indicates localization in vesicles or endosomes. Co-expression experiments revealed a precise co-localization of MuSK and Cl-II6. In addition we could show that the protein is localized at the neuromuscular junction. Ongoing experiments con- centrate on the functional analysis of the newly identified MuSK binding protein with particular emphasis on its potential role as a protein involved in MuSK trafficking. Abstracts Transport Machineries 98 ª 2007 The Authors Journal compilation ª 2007 FEBS B1-9 Motor protein KIFC5A and its interacting proteins Nubp1&Nubp2 are involved in the regulation of centrosome duplication A. Christodoulou 1 , C. W. Lederer 1 , T. Surrey 2 , I. Vernos 3 and N. Santama 1 1 University of Cyprus, Nicosia, CYPRUS, 2 EMBL, Heidelberg, GERMANY, 3 Centre of Genomic Regulation, Barcelona, SPAIN Inhibition of motor protein activity is linked with defects in the for- mation of poles in the mitotic spindle but the molecular mecha- nisms underlying the functional relationship between motor activity and centrosome dynamics are unclear. We characterised KIFC5A, a minus-end-directed mouse kinesin-like protein, highly expressed in dividing cells. It is nuclear in interphase, localises to the centre of spindle asters at the beginning of mitosis, and to spindle MTs in later phases. Overexpression of KIFC5A in mouse cells causes the formation of non-separated MT asters and mitotic arrest in a pro- metaphase-like state. KIFC5A knockdown partly rescues the phe- notype caused by monastrol inhibition of plus-end-directed motor Eg5, indicating that it is involved in the balance of forces determin- ing bipolar spindle assembly and integrity. Silencing of KIFC5A results in centrosome amplification detectable throughout the cell cycle. Supernumerary centrosomes arise primarily by reduplication and partly from cytokinesis defects. They contain duplicated centri- oles and have the ability to organise MT asters, resulting in the for- mation of multipolar spindles. KIFC5A interacts with nucleotide- binding proteins 1 and 2 (Nubp1, Nubp2), which have extensive sequence similarity to prokaryotic division-site-determining protein MinD. Nubp1&Nubp2 also interact with each other. Knockdown of Nubp1 or double knockdown of Nubp1&Nubp2 both pheno- copy the KIFC5A silencing effect, implicating KIFC5A and the Nubps in a regulatory pathway involved in the control of centro- some duplication. B1-10 Which structural determinants make kinesin processive? S. Adio 1,2 , B. Ebbing 2 , J. Jaud 2 , M. Rief 2 and G. Woehlke 2 1 National Institute for Medical Research, London NW7 1AA, UK, 2 Technical University of Munich, Munich, GERMANY Kinesin motor proteins transport cargo through the cell. Conven- tional Kinesin-1 motors travel processively along microtubules by taking hundreds of successive ‘hand-over-hand’ steps. They are composed of two identical subunits, each consisting of a motor domain and a neck/stalk. Despite extensive studies, structural ele- ments responsible for the accurate coordination between the two subunits remain unknown. To identify these elements, we have constructed a chimeric motor with the motor domain of Kinesin-1 and a neck/stalk of a non-processive Kinesin-3. We found that this novel kinesin behaves qualitatively as conventional Kinesin-1. It moves processively in single molecule fluorescence assays and exerts up to 3 pN force in optical trapping experiments. However, reduced velocities and run lengths at a variety of loads indicate that the unconventional neck domain hinders the diffusive search for the next binding site. In the reverse chimera (motor domain of non-processive Kinesin-3 and neck/stalk of Kinesin-1) the Kinesin- 1 neck allows sequential ADP release from the partner heads upon microtubule binding. But this motor was unable to make successive steps. Our observations suggest that kinesin processivity requires two independent elements. One, located in the neck/stalk region, ensures coordinated ADP release from the motor domains. The other, located in the motor domain itself, allows successive ‘hand- over-hand’ stepping by coordinating the timing of events between the two motor domains. B1-11 Effects of kinesin motor domain structure on kinesin activity N. Kalchishkova, E. Unger and K. J. Bo ¨ hm Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena, GERMANY Kinesin-microtubule interaction results in generation of motility, essential for organelle transport and cell division. Each kinesin molecule reveals a distinct globular motor domain, a neck-linker and a neck, a stalk and a cargo-binding tail domain. The force- generating motor domain comprises both the microtubule-binding site and the ATP-hydrolysis centre. Kinesin binding to microtubules, known to promote kinesin AT- Pase activity, induces the nucleotide-binding pocket to be closed and initializes ATP hydrolysis followed by subsequent conforma- tional changes, triggering the directed movement of the motor pro- tein (Naber et al. Science 30, 798). So far, the molecular mechanisms of kinesin-microtubule interaction cannot be com- pletely understood. The a5-helices of kinesin-1 and Eg5 and their neighbouring loops are important surfaces undergoing nucleotide- dependent rearrangements to interact with microtubules (Turner et al. J. Biol. Chem. 276, 25496). Therefore, we expressed both human neuronal kinesin-1 and mitotic Eg5 constructs of different length with changes mainly in sequences following the a5 helix to find the minimal length of the motor domain still allowing binding to microtubules, ATP hydrolysis, and finally motility generation. In addition, chimeric constructs of fast (kinesin-1) and slow (Eg5) motors have been expressed and characterized to understand velo- city regulation. We demonstrated that kinesin-1 and Eg5 constructs terminated at a5 helix and its neighbouring loops do not reveal a microtubule- promoted ATPase activity. Thus, structures next to these regions are considered to have an important function in conformational modulation of motor domain during the ATP hydrolysis cycles and motility generation. B1-12 Quality control during the asse mbly of myosin-V dependent translocation complexes A. Heuck 1 ,T.Du 2 , R. Jansen 2 and D. Niessing 1 1 GSF & Gene Center Munich University, Munich, GERMANY, 2 Gene Center Munich University, Munich, GERMANY Motor protein-dependent transport of cargo is a basic cellular function. To exert their motile activity, the molecular motors kine- sin and myosin must dimerize. Surprisingly little is known about how such molecular motors assemble with other proteins into func- tional translocation complexes. Recent studies on a minus-end directed myosin, i.e. type VI myosin, show that this motor only di- merizes upon complex assembly. However, for plus-end directed myosins, which constitute the vast majority of myosin motors, we know very little about the mechanisms controlling the assembly of their translocation complexes. Available data suggest for these my- osins a constitutive and unregulated interaction of their C-terminal tails with cargo complexes. We report a study involving biochemical, biophysical, and in vivo experiments on the assembly of a yeast translocation complex with plus-end directed myosin type V. Our results establish that myosin dimerization significantly increases its affinity for its interaction partner of the cargo complex and stabilizes the resulting complex. Thus, myosin dimerization is required for efficient binding to the core complex. Since only dimeric myosins move processively along actin filaments, these observations reflect a quality control step for the selective assembly of functional translocation complexes. Our finding contrasts the previously suggested simple interaction of the myosin C-terminal tail with cargo complexes. Transport Machineries Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 99 B1-13 A functional multivesicular body pathway is required for clearance of protein aggregates by autophagy Filmonenko M. 1 , Raiborg C. 1 , Stuffers S. 1 , Yamamoto A. 2 , Brech A. 1 , Stenmark H. 1 and Simonsen A. 1 1 Dept. of Biochemistry, Rikshospitalet-Radiumhospitalet HF, N-0310 Oslo, NORWAY 2 Dept. of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA The ESCRT-III subunit CHMP2B/Vps2b was recently found to be mutated in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These diseases are characterized by abnormal intracellular ubiquitin positive protein deposits in affected neurons. The ESCRTs, first identified in yeast as vps class E mutants, have proven important for recognition of ubiquitinated endocytosed integral membrane proteins and their sorting into the intralumenal vesicles of the multi-vesicular body (MVB) and subse- quent degradation in the lysosome/vacuole. We here present evi- dence that siRNA-mediated depletion of the ESCRT III subunit Vps24 leads to accumulation of large ubiquitin-positive cytoplasmic protein aggregates, which also contain p62/Sequestosome-1 and Alfy (autophagy-linked FYVE protein), but are devoid of the early endo- some marker EEA1. We show that p62-containing amphisomes (positive for GFP-LC3 and CD63) do form in Vps24 depleted cells, but that lysosomal degradation of p62 and LC3 is inhibited/delayed. Vps24 is also required for efficient clearance of HttQ103 aggregates in a cell-based model of Huntingtons disease. Taken together, our data indicate that efficient autophagic clearance of ubiquitinated proteins requires functional MVBs and provide a possible explan- ation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations. B1-14 Screen for new factors affecting function of ubiquitin ligases, human Nedd4 and yeast Rsp5, in yeast S. cerevisiae P. Kaliszewski 1 , M. Stawiecka-Mirota 1 , D. S. Haines 2 and T. Zoladek 1 1 Institute of Biochemistry and Biophysics PAS, Warsaw, POLAND, 2 Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA Human Nedd4 and yeast Rsp5 are homologous ubiquitin ligases that contain a catalytic Hect domain, a C2 domain and multiple WW domains interacting with proteins. Previous studies indicated that NEDD4 gene can be efficiently expressed in yeast, however, can not replace RSP5. Expression of NEDD4w4 with mutation inactivating WW4 domain of Nedd4 ligase is toxic for yeast cells. In this study we analyzed the survival of cells overproducing Nedd4w4 and showed that it causes inhibition of cell growth, not the lethality. Multicopy genomic library was screened for genes which suppress this growth defect. Five genes were isolated among them ATG2 which is involved in autophagy. Using tester strain pho8D60 we showed that overexpression of NEDD4w4 or ATG2 genes results in increase of autophagy in growth conditions and the effect is additive. These results indicate that expression of nedd4w4 possibly results in defects of proteasomal protein degra- dation and induction of autophagy may help to overcome this problem. B1-15 The lysosomal targeting of the membrane protein p40 is mediat ed by a dileucine signal situated in its C-terminal tail M. Boonen, G. Cuvelier, R. Castro, I. Hamer and M. Jadot URPHYM, FUNDP, Namur, BELGIUM Transport of newly synthesized lysosomal membrane proteins from the TGN to the lysosomes is due to the presence in their cytoplas- mic domains of specific signals which are recognized by clathrin- associated adaptor complexes. p40, a predicted multispanning pro- tein of 372 amino acids localized to the lysosomal membrane, con- tains four putative lysosomal sorting motifs in its sequence: three of the YXXF-type (Y 6 QLF, Y 106 VAL, Y 333 NGL) and one of the dileucine-type (EQERL 360 L 361 ). Mutations of the critical residues of these motifs, tyrosine or leucine, revealed that the EQERLL motif was implicated in the sorting of p40. Confocal microscopy analyses, performed in transfected HeLa cells, showed that p40- GFP was mis-targeted to the plasma membrane when its dileucine motif was disrupted. This result was confirmed by cell surface bio- tinylation studies which allowed us to estimate that approximately 50% of the p40 dileucine mutants (without GFP) were associated with the plasma membrane. We also observed that, in self-forming Percoll density gradients, the dileucine mutants were excluded from fractions containing mature lysosomes. Taken together, our results show that the sorting of p40 from the TGN to the lysosomes is directed by the dileucine EQERL 360 L 361 motif situated in its C-ter- minal tail. B1-16 Elongation arrest activity of SRP is essential for efficient translocation of proteins into the ER in mammalian cells C. Mary 1 *, A. K. K. Lakkaraju 1 *, A. Scherrer 1 , A. E. Johnson 2 and K. Strub 1 1 University of Geneva, Geneva, SWITZERLAND, 2 A&M University System Health Science Center, College Station, TX, USA The signal recognition particle (SRP) is responsible for co-transla- tional translocation of proteins bearing signal sequence into the endoplasmic reticulum (ER). In wheat germ translation system, SRP causes an arrest in the elongation (EA) of nascent chains pre- senting signal sequence and efficiency of their translocation is dependent on this arrest. We have identified a five amino acid-long motif in SRP14 required to confer EA activity to SRP. Particles comprising the defective SRP14 protein (A5), no longer arrest elon- gation of the nascent chain and have reduced translocation effi- ciency. To examine the significance of this function for protein secretion in mammalian cells, we depleted endogenous SRP14 using RNAi and replaced it with the expression of wild type or mutant GFP-14. Although the expression of both, GFP-14 and GFP-14A5, restored cellular SRP levels to normal, the translocation efficiency of reporter proteins was specifically diminished in cells expressing GFP-14A5. The defect could be rescued by the addition of aniso- mycin, which slows down nascent chain elongation. These results demonstrate the physiological relevance of EA for efficient target- ing in mammalian cells. Protein translocation into the ER is ineffi- cient at normal cellular translation elongation rates presumably, because nascent chains become too long during the time span required for SRP to target nascent chains successfully to the trans- locon. The selective slow down of translation elongation by SRP is therefore required to optimize protein translocation efficiency in mammalian cells without hampering continued rapid translation of other cellular proteins. *equal contribution. Abstracts Transport Machineries 100 ª 2007 The Authors Journal compilation ª 2007 FEBS B1-17 Regulation of vesicle trafficking by protein S-nitrosylation Y. Daaka Medical College of Georgia, Augusta, GA, USA The guanosine triphosphatase (GTPase) dynamin regulates endocy- tic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We repor- ted that dynamin, which interacts with nitric oxide (NO) synthase, is S-nitrosylated at a single cysteine residue. C607. S-nitrosylation increases dynamin self-assembly and GTPase activity, and facili- tates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO generating cells, expression of dynamin C607A, like the GTPase-deficient domin- ant-negative K44A dynamin, inhibits active receptor internalizat- ion. More recent results implicate the dynamin S-nitrosylation in infectious particle uptake. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. We will discuss the general NO-dependent mechanism by which the trafficking of plasma membrane vesicles may be regulated, and the specific idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry. B1-18 The role of signaling in control of bi-directional ER to Golgi trafficking V. Gupta and D. Stephens Dept. of Biochemistry, University of Bristol, UK Our research is aimed at understanding the function of the early secretory pathway in mammalian cells. While we now have a signi- ficant amount of knowledge regarding the individual molecules involved, we now need to develop this to an understanding of the system as a whole. We have developed live cell imaging assays to analyze the individual movements of structures in transit between the ER and the Golgi, the first two compartments of the secretory pathway. Movement of these transport carriers occurs in a stop- start fashion with frequent bidirectional oscillatory, as well as lon- ger range, movements. This behaviour is due to regulation of the binding to, and/or regulated activity of, opposing motor proteins- in general terms, dynein (along with dynactin) moves cargo towards the Golgi, while kinesin moves cargo away from it. The function of these motors is also controlled by protein phosphoryla- tion, acting at least in part through a protein kinase A-dependent pathway. We are studying the association of dynactin with carriers in transit to the Golgi and the regulation of bidirectional motility by protein phosphorylation by imaging GFP-tagged forms of var- ious ER-Golgi markers. We have established an experimental sys- tem that allows us to image the movement of ER-to-Golgi transport carriers with extremely high spatial and temporal resolu- tion. Application of 2D-Gaussian fitting models to the data allows precise determination of object position. This should ultimately allow us to determine motor step size, which will give key informa- tion regarding the type of motors associated with these structures. It will also lead to the detailed analysis of the dynamics of trans- port carriers operating between early secretory pathway and help in deciphering the mechanism of their regulation. B1-19 Molecular analysis of YIP1 family protein function C. Chen and R. N. Collins Cornell University, Ithaca, NY, USA The YIP1 family is a group of integral membrane proteins with the capability of binding to dual prenylated Rab GTPases. These proteins are functionally conserved from yeast to humans, and, in S. cerevisiae, comprise two essential proteins Yip1p and Yif1p, and two non-essential proteins Yip4p and Yip5p. Although their mode of action remains unclear at the molecular level, phenotypic analy- sis of yip1 and yif1 mutant cells reveal blockage in early stages of membrane traffic. We have previously shown that Yip1p is neces- sary for biogenesis of ER derived COPII coated vesicles both in vivo and in vitro (1). In our current study, we utilized serial trunca- tion and site-directed mutagenesis to isolate alleles of Yif1p and Yip1p which abrogate their Rab-interacting capabilities. Surpris- ingly, our results suggest that the YIP1 family proteins might have a function independent of Rab proteins, and present data to sug- gest a possible role in organelle shape. Reference 1. Heidtman M, Chen CZ, Collins RN, Barlowe C. A role for Yip1p in COPII vesicle biogenesis. J Cell Biol. 2003 Oct 13;163 (1) :57–69. B1-20 GTRAP3-18 serves as a negative regulator of Rab1 in protein transport and neuronal differentiation S. Maier 1 , V. Reiterer 1 , A. M. Ruggiero 2 , J. D. Rothstein 3 , H. H. Sitte 1 and H. Farhan 1 1 Institute of Pharmacology, Medical University, Vienna, AUSTRIA, 2 Department of Pharmacology, Vanderbilt University, Nashville, TN, USA, 3 Departments of Neuroscience and Neurology, Johns Hopkins University, Baltimore, MD, USA GTRAP3-18 is an ER localized protein belonging to the prenylat- ed rab-acceptor-family interacting with small Rab GTPases. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 inhibits Rab1, which is involved in ER-to-Golgi trafficking. The effects on the early secretory pathway were: reduction of the rate of ER-to-Golgi transport in HEK293 cells, inhibition of cargo concentration of the EAAC1 glutamate transporter into transport complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited neurite out- growth in CAD cells. Finally, we hypothesized that expression of GTRAP3-18 in the brain should be lower at stages of active syn- aptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 was lower in P0 rat brains com- pared to E17 rat brains. Transport Machineries Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 101 B1-21 The signal peptide of LCMV GP-C is required for glycoprotein maturation and virus infectivity S. Schrempf 1 , M. Froeschke 1 , T. Giroglou 2 , D. von Laer 2 and B. Dobberstein 1 1 Zentrum fu ¨ r Molekulare Biologie Heidelberg (ZMBH), Heidelberg, GERMANY, 2 Georg-Speyer-Haus (GSH), Frankfurt, GERMANY Signal peptides direct nascent secretory and membrane proteins to the endoplasmic reticulum (ER) and are usually cotranslationally cleaved off from the preprotein by signal peptidase. Insertion of the lymphocytic choriomeningitis virus (LCMV) precursor glyco- protein C (pGP-C) into the ER membrane is mediated by an unu- sual signal peptide (SP GP-C ). It is longer than average SPs comprising an extended hydrophilic N-terminal (n) region inclu- ding a myristoylation site and two hydrophobic (h) regions. After cleavage by signal peptidase SP GP-C was found to accumulate in cells and virus particles. In the present study, we identified the LCMV SP GP-C as an essential component of the viral GP complex. By using several deletion and point mutants we investigated signal sequence require- ments for GP-C biosynthesis, transport to the cell surface and viral infectivity. Our results show, that only one SP GP-C h-region is nee- ded for membrane insertion of pGP-C, whereas both h-regions are required for GP-C processing and cell surface expression. Further- more, the n-region of the SP GP-C and the myristoylation are found to be essential for viral infectivity, most likely for pH-dependent fusion with the host cell membrane. Thus different regions of LCMV SP GP-C fulfil essential functions in GP-C maturation and virus infection. B1-22 Different domains of GOPC regulate its Golgi localization I. Todros 1,2 , E. Kurzejamska 1,3 , A. Matysiak 1,3 and M. Milewski 1 1 Institute of Mother and Child, Warsaw, POLAND, 2 Agricultural University SGGW, Warsaw, POLAND, 3 Warsaw University, Warsaw, POLAND Golgi-associated PDZ and coiled coil motif-containing protein (GOPC) is a peripheral Golgi protein involved in vesicular traffick- ing of many integral membrane proteins, including CFTR, a pro- tein defective in cystic fibrosis. The exact role of GOPC in post- Golgi trafficking is not known, although it has been suggested that it may function as an adaptor protein, participating in transport from Golgi to lysosomes. To identify the amino acids sequences responsible for subcellular localization of GOPC, a series of dele- tions encompassing different structural domains was introduced into the protein sequence. Immunofluorescence analysis of the sub- cellular distribution of these GOPC variants in transfected epithe- lial cells showed that the second coiled-coil domain (CC2) is required for Golgi localization, with adjacent second conserved region (CR2) also contributing to the efficiency of Golgi targeting. Additionally, increased Golgi localization of the GOPC variants devoid of the C-terminal PDZ domain suggested that Golgi distri- bution of GOPC is negatively regulated by the PDZ-mediated interactions. This was further supported by the fact that overex- pression of CFTR, a protein interacting with GOPC through its PDZ domain, substantially reduced Golgi localization of GOPC. Thus, different domains of GOPC seem to contribute to its subcel- lular distribution. While the coiled-coil-mediated interactions ensure efficient targeting to the Golgi, the PDZ-based mechanism, being responsible for binding a cargo protein, contributes to the subsequent detachment of the whole complex from the trans-Golgi membrane. Acknowledgement: Supported by grant PBZ/KBN/122/P05/01- 06. B1-23 Role of the multimolecular conserved oligomeric Golgi complex in intra-Golgi trafficking and glycosylation disorders V. Lupashin, A. Shestakova, E. Suvorova, R. D. Smith and O. Pavliv University of Arkansas for Medical Sciences, Little Rock, AR, USA Large oligomeric multiprotein complexes direct membrane trafficking in eukaryotic cell. One class of traffic regulators, vesicle tethering fac- tors, mediate the initial loose tethering of transport vesicles to their target membranes. The peripheral membrane conserved oligomeric Golgi (COG) complex directs retrograde intra-Golgi membrane traf- ficking and ensures proper Golgi functions. Mutations in COG sub- units cause congenital glycosylation disorders in humans, suggesting that COG is necessary for the correct sorting of glycosyltransferases, which create a variety of developmentally important glycans. The COG complex consists of two lobes, Lobe A (COG1-4) and Lobe B (COG5-8). Rapid knock-down of Lobe A subunits induces accumu- lation of intra-Golgi vesicles and Golgi ribbon breakdown, while the knock-down of Lobe B subunits COG6 and COG8 does not change Golgi morphology. The COG complex functionally communicates with a subset of Golgi SNAREs and Rab proteins. Yeast COG com- plex interacts with the SNARE domain of Sed5p and preferentially binds to the Sed5p-containing SNARE complex. Yeast two hybrid assay identifies mammalian Cog4p and Cog6p subunits as binding partners of the mammalian Golgi t-SNARE Syntaxin5. FRET assay reveals direct in vivo interaction between Syntaxin5 and the COG complex. Knock-down of the COG complex results in decreased Golgi SNARE mobility, accumulation of free uncomplexed form of Syntaxin5 and decrease in steady-state level of intra-Golgi SNARE complexes. In summary, the COG complex acts to promote forma- tion and/or stabilization of the intra-Golgi SNARE complex. Acknowledgements: Supported by grants from the NSF (MCB- 0234822) and Mizutani Foundation for Glycoscience. B1-24 Cargo passage through the golgi apparatus induces golgi lumenal Ca 2+ fluctuations M. Micaroni 1 , K. Bianchi 2 , R. Rizzuto 2 , G. Perinetti 1 , A. Spaar 1 , D. Di Giandomenico 1 , A. Luini 1 and A. A. Mironov 1 1 Consorzio ‘‘Mario Negri Sud’’, Santa Maria Imbaro, ITALY, 2 Uni- versity of Ferrara, Ferrara, ITALY Recent evidence has highlighted the functional importance of Ca 2+ in intracellular trafficking, and Ca 2+ increases could act downstream of the SNAREs, proteins that are involved in the fission/fusion processes that occur during cargo progression through the Golgi apparatus. Indeed, C a 2+ increases the rate of SNARE-mediated membrane fusion via C a 2+ ion channel association with the SNARE complex. Our work- ing hypothesis is based on the fact that the fusion events, that occur during cargo progression from the Golgi apparatus to the trans- Golgi network (TGN), involve redistribution of the Ca 2+ stored in the lumen of the Golgi cisternae that mediates the recruitment of reg- ulatory proteins involved in the final steps of SNARE-regulated fusion. Here we have examined whether the Ca 2+ concentrations near the Golgi apparatus and inside the trans-Golgi lumen change during cargo progression through the Golgi. Using fluorescence res- onance energy transfer (FRET) with HeLa cells stably transfected with galactosyl transferase-Yellow Chameleon 3.3 (GT-YC3.3) in vivo and in fixed samples, we have seen Ca 2+ leakage in the trans- Golgi region from the lumen of Golgi cisternae in VSV-infected cells during the passage of the viral VSVG protein. At the same time, in HeLa cells transfected with sialyl transferase-aequorin (ST-Aeq), we have confirmed this release of Ca 2+ from the lumen of the Golgi cisternae. Moreover, we have seen an increased frequency of cyto- solic Ca 2+ spikes using FURA-2. These data suggest that the redis- tribution of Ca 2+ during the movement of cargo proteins through the Golgi apparatus has a significant role in intra-Golgi transport. Abstracts Transport Machineries 102 ª 2007 The Authors Journal compilation ª 2007 FEBS B1-25 Direct interaction of the C-terminus of TRPC6 with Rab9 targets TRPC6 to the trans-Golgi network S. Cayouette and G. Boulay Universite ´ de Sherbrooke, Sherbrooke, PQ, CANADA TRPC proteins are implicated in Ca 2+ entry following activation of Gq-protein coupled receptors. We previously showed that the inser- tion of TRPC6 into the plasma membrane (PM) is regulated by hor- monal stimulation. Since small G proteins of the Rab family are involved in vesicle trafficking and fusion, we assessed the intracellu- lar trafficking of TRPC6 with different Rab dominant negative (DN) mutants. TRPC6 activation was measured in HEK293 cells transiently co-expressing either Rab11DN (blocks transport from the TGN and recycling endosome to the PM) or Rab9DN (blocks fusion of late endosomes with the TGN). Rab9DN significantly enhanced carbachol-induced Ca 2+ entry through TRPC6 activation, whereas Rab11DN completely abolished its activity. Rab11 and Rab9 have no significant effect on TRPC6 activity. GST-pulldown assay showed that Rab9 and Rab9DN interact directly with the C- terminus of TRPC6. These interactions occur also in vivo, since Rab9 and Rab9DN co-immunoprecipitate with TRPC6. However, neither Rab11 nor Rab11DN interact in vivo with TRPC6. Immuno- fluorescence microscopy and cell-surface biotinylation assays showed that Rab9DN enhances the amount of TRPC6 at the cell surface and Rab11DN causes an intracellular retention of TRPC6. We conclude that TRPC6 transits through the TGN and that Rab9 is directly involved in the process. Moreover, TRPC6 is redirected to the PM in a Rab11-dependent fashion without involving an interac- tion with TRPC6. Supported by CIHR and FMCQ. B1-26 Role of Casein Kinase 2 in the early steps of CFTR biogenesis C. M. Farinha 1,2 , L. Pissarra 1 and M. D. Amaral 1,2 1 Faculty of Sciences, University of Lisboa, Lisboa, PORTUGAL, 2 Cent Hum Genet, Nat Inst Health, Lisboa, PORTUGAL Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR), the product of the gene which is mutated in CF, func- tions as a Cl - channel at the apical membrane of epithelial cells. The most frequent mutation (F508del) causes retention of its protein product in the endoplasmic reticulum as a core-glycosyl- ated intermediate that is rapidly degraded. Thus, F508del-CFTR fails to traffic to the plasma membrane. It was recently shown that Casein Kinase 2 (CK2) binds wt-CFTR near the F508 resi- due and phosphorylates serine residue at position 511. Deletion of F508 abrogates this residue-dependent interaction [1]. Our aim here was to identify whether CK2 interaction affects CFTR biogenesis, turnover and processing. BHK cells were stably transfected with wt- or F508del-CFTR in which S511 was sub- stituted by alanine (S511A) or aspartate (S511D). Pulse-chase experiments followed by CFTR immunoprecipitation were per- formed in these lines, showing that substitution of S511 does not affect the turnover or processing of either wt- or F508del- CFTR. The effect of CK2 inhibition on the turnover and pro- cessing of CFTR showed that: 1) steady-state levels of CFTR are reduced. 2) turnover of wt-CFTR (but not F508del-CFTR) is increased and 3) processing of wt-CFTR is decreased. Our data suggest a putative stabilizing role for CK2 upon wt-CFTR, not dependent on the charge that is added by the kinase, so it is probably indirect. Acknowledgement: Work supported by CIGMH and POCTI/ SAU/MMO/58425/2004 grant (FCT, Portugal). Reference 1. Treharne et al. (2007) J Biol Chem (epub). B1-27 Checkpoints in the traffic pathways of Tyrosinase Related Proteins in melanoma cells G. Negroiu Institute of Biochemistry, Bucharest, ROMANIA Tyrosinase Related Proteins-(TRPs) are the main regulators of melanin synthesis in normal and pathological pigmentation and immune targets in melanoma, glioma and autoimmune depigmen- tation. TRP-2 has a significant role in intrinsic drug resistance of tumor phenotypes. TRPs are all type I transmembrane N-glyco- proteins which follow the common biosynthetic secretory pathway. TRP-polypeptide folding and early N-glycan processing are accom- plished in ER. TRPs move than via the Golgi complex to the TGN, where are sorted and transported to the membrane of mel- anosomes, the organelles for melanin synthesis and temporal deposition. Abnormalities in the ER or post-ER events of the intracellular processing of tyrosinase and TRP-1 result in their mis- routing or/and degradation and have been investigated in different pigmentary diseases and types of malignancy. TRP-2 biosynthesis was analyzed for the first time in our studies using pharmacologi- cal agents as modulators of intraluminal pH together with TRP-1 as a reporter molecule. We found that TRP-2 is sorted and traf- ficked in the early secretory pathway with a cargo which does not include TRP-1, supporting the concept of a selective ER-Golgi transport; post-Golgi, unlike TRP-1, TRP-2 intersects the endocy- tic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These findings demon- strate that, in spite of the high degree of structural homology, the traffic pathways of TRPs are individually regulated. TRPs face dis- tinct ‘‘check points’’ along their biosynthetic journey in order to become functional proteins. B1-28 Impact of progesterone on cytokine-stimulated NF-jB signaling A. C. Vidaeff University of Texas Houston Medical School, Houston, TX, USA Objective: A key event in the pathways leading to preterm labor may be the activation of nuclear factor-jB (NF-jB). Anti-inflam- matory agents, such as the corticosteroids, inhibit the activation of NF-jB. We proposed to investigate the effects of progesterone (P) pretreatment on cytokine-stimulated activation of NF-jB. Study design: HeLa cells were pretreated with 10 -7 M progester- one for 24 h and exposed to 1 ng/ml IL-1b for 1 hour. Nuclear and cytosolic extracts were subjected to Western analysis using anti-p65 and anti-IjBa antibodies. Densitometric data (n =5) were compared using Kruskal–Wallis test. Results: Pretreatment with P interfered with IL-1b-induced inhibi- tory protein-jBa (IjBa) degradation. However, P pretreatment resulted in a significant decrease in p65 in the cytoplasm. Pretreat- ment with P did not reduce the amount of nuclear p65 and did not interfere with nuclear translocation of p65. Conclusion: Our observations suggest that any possible role played by P in preterm labor prevention is not exerted through anti-inflammatory mechanisms of NF-jB downregulation. Transport Machineries Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 103 B1-29 Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function M. Setou NIPS and MITILS, Machida, JAPAN Microtubules function as molecular tracks along which motor pro- teins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamyla- tion, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we ana- lyzed ROSA22 mice that lack functional PGs1, a subunit of alpha- tubulin-selective polyglutamylase. In wild-type mice, polyglutamyl- ated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha- tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule- associated proteins and motor proteins, including kinesins, to micro- tubules purified from ROSA22-mutant brain. Of the kinesins exam- ined, KIF1A was less abundant in neurites from ROSA22 mutants in vitro and in vivo. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesi- cles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission. Further investigation of the role of polyglutamylation in vivo will be dis- cussed in this presentation. B1-30 Yif1B is implicated in the dendritic localisation of the serotonin 5-HT1A receptor J. Masson 1,2 , S. Al Awabdh 1,2 , D. Carrel 1,2 , C. Borg-Capra 3 , J. Laine ´ 2,4 , M. Hamon 1,2 , M. B. Emerit 1,2 and M. Darmon 1,2 1 INSERM U677, Paris, FRANCE, 2 Universite ´ Pierre et Marie Curie, Paris, FRANCE, 3 Hybrigenics SA, Paris, FRANCE, 4 Dept Physiologie, UPMC, Paris, FRANCE The 5-HT1A receptor is a dendritic receptor implicated in the con- trol of brain (5-HT) neurotransmission. Evidence has been reported that the short C-terminal domain of the rat 5-HT1AR plays a crucial role in receptor targeting from the endoplasmic reticulum to the plasma membrane. We used this 17 amino acid region as a bait in a yeast two-hybrid screen and identified Yif1B as a new partner of 5- HT1AR. The physical interaction between the receptor and Yif1B was fully confirmed in GST pull down experiments with 5-HT1AR C-tail on rat brain extracts. Yif1B is the ortholog of the yeast Yif1p protein ( Yip1 Interacting Protein, implicated in the vesicular traffic between the endoplasmic reticulum and the Golgi apparatus, Matern et al. 2000). Yif1B is highly expressed in the rat brain as shown by northern and western blot analyses. Immunolabeling with specific polyclonal antibodies showed that in transfected cell lines Yif1B was confined to an intracellular vesicular compartment partially overlap- ping the Golgi apparatus, in concordance with the role of its yeast Yif1p ortholog. Electron microscopy visualization of endogenous Yif1B protein in rat brain tissue sections confirmed this Golgi locali- sation. Finally, siRNA-induced inhibition of endogenous Yif1B expression in neuronal primary cultures prevented the addressing of 5-HT1AR in distal portions of dendrites. All these results support the hypothesis that Yif1B plays a role in the dendritic localisation of the 5-HT1A receptor in brain neurons. B1-31 Alpha-Syntrophin interacts with Kidins220 and Protein Kinase D in neural cells L. Sa ´ nchez Ruiloba, A. Higuero, R. Martı ´ n Jean-Mairet, M. Rodrı ´ guez-Martı ´ nez, F. Portillo and T. Iglesias Insituto de Investigaciones Biomedicas, Madrid, SPAIN Protein kinase D1 (PKD1) belongs to a novel family of diacylglyc- erol (DAG)-stimulated Ser/Thr kinases, constituted by two more members, PKD2 and PKD3. We have previously identified the C terminus of the different PKDs that constitutes a PSD-95, Dlg, ZO-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of Kinase D-interact- ing substrate of 220-kDa (Kidins220) surface localization. Ki- dins220 is a neural membrane protein identified as the first substrate of PKD1. PKD1 controls Kidins220 transport by the interaction with a PDZ protein. In an effort to identify that PDZ protein we performed yeast two hybrid screenings using as baits the PDZ-binding motifs of Kidins220 and PKD1. We found a pos- itive clone containing the complete coding sequence of the PDZ protein a-syntrophin that strongly interacted with both baits. a- syntrophin has been described as a Kidins220 binding partner that plays an important role in the regulation of Kidins220 localization during neuromuscular junction differentiation. Our results show that PKD1 phosphorylation of its PDZ-binding motif regulates the interaction with a-syntrophin in yeast and that Kidins220, PKD1 and a-syntrophin form ternary complexes in neural cells. These data suggest that the interaction of a-syntrophin with Kidins220 and PKD1 could be relevant for the control the PKD1 exerts on Kidins220 transport. B1-32 Cellular control of trafficking and activity of perforin, a key regulator of immune homeostasis I. Voskoboinik Peter MacCallum Cancer Centre, East Melbourne, AUSTRALIA Perforin (PRF) is an essential cytolytic pore-forming protein stored in the secretory granules of cytotoxic lymphocytes (CL). CLs kill virus infected and transformed cells through the granule-mediated pathway, where PRF synergises with serine proteases, granzymes, to deliver the lethal hit. In humans, congenital PRF deficiency cau- ses a fatal disorder of immune system, FHL or, in milder cases, it affects tumour immune surveillance, which results in haematologi- cal cancers (1–3). Despite the essential role of PRF in immune sys- tem, its mechanism of action and the cell biology remain largely unknown. Activated CLs synthesize a large amount of PRF, and a key question in CL biology is how these cells protect themself from the self-lysis by endogenous PRF. We have found that the extreme C-terminal region of PRF encodes structural and trafficking motifs which, acting in concert, are responsible for the inhibition of its cytotoxic activity and, at the same time, allow PRF trafficking to cytotoxic granules. There, PRF acquires an activated state through modifications of its C-terminal tail. However, the acidic pH in the lumen of the organelle prevents PRF from exerting its cytotoxicity prior to its release outside the cell into the immune synapse. Thus, we unravelled the interplay between finely tuned trafficking path- ways and post-translational activation mechanisms of PRF, which ensures its safe delivery and storage in the secretory granules. References 1. Voskoboinik et al. (2006) Nature Rev Immunol 6, 940. 2. Voskoboinik et al. (2004) J Exp Med 200, 811. 3. Voskoboinik et al. (2005) Blood 105, 4700. Abstracts Transport Machineries 104 ª 2007 The Authors Journal compilation ª 2007 FEBS B1-33 Regulation of intracellular trafficking plays a pivotal role in tumor pathogenesis J. Go ¨ ttert Max-Delbru ¨ ck-Centrum fu ¨ r Molekulare Medizin, Berlin, GERMANY Intracellular trafficking as a fundamental cellular process needs to be tightly regulated, otherwise disturbances can lead to a variety of disorders and diseases. Glycans play a pivotal role in protein sorting, there synthesis proceeds in a hierarchical manner along the secretory pathway. Mechanisms that alter the intracel- lular localization of glycosyltransferases and glycosidases may lead to an alternative glycan repertoire of cells. In addition, gly- can linkage defects are associated with pathogenetic events in hereditary and acquired human diseases. Here, we refer to EBAG9 that has been identified as a primary estrogen respon- sive gene. High expression levels of the gene product have been recorded in several carcinomas, suggesting a tumor-promoting role of the protein. EBAG9 is a ubiquitously expressed Golgi protein with a peripheral membrane attachment. It was shown to induce expression of the truncated O-linked-glycans Tn and TF at the plasma membrane of non-secretory epithelial cell lines. In general, Tn- and TF are extensively expressed on the cell sur- faces of diverse tumor tissues and they are thought to be involved in tumor cell adhesion, invasion and metastasis. How- ever, the mechanism underlying their generation remains to be elucidated. Here, we localize the EBAG9-imposed alterations to the early biosynthetic pathway and provide a functional link between hormone-dependent gene expression, tumor-associated O-linked glycan deposition, vesicle transport, and tumor patho- genesis. B1-34 Enhancement of the fungicidal activity of amphotericin B by allicin, an allyl sulfur compound from garlic A. Ogita, M. Ogita, K. Fujita and T. Tanaka Osaka City University, Osaka, JAPAN Amphotericin B (AmB) is a representative antibiotic for the control of serious fungal infections, and its fungicidal activity was greatly enhanced by allicin, an allyl sulfur compound from garlic. In Sac- charomyces cerevisiae, allicin was only slightly effective or ineffective in enhancing the fungicidal activities of other antibiotics causing plasma membrane damage. AmB increased plasma membrane per- meability in favor of potassium efflux from intact cells, but AmB- induced plasma membrane damage was unaffected or attenuated in the presence of allicin. At a lethal concentration, AmB additionally induced vacuole membrane damage so that the organelles were vis- ible as small discrete particles. AmB caused a serious structural dam- age to the yeast vacuole even at a nonlethal concentration in combination with allicin that interferes with ergosterol trafficking from plasma membrane to the organelles. Allicin could also decrease the minimum fungicidal concentration (MFC) of AmB against the pathogenic fungus Candida albicans to less than 12.5%, and against Aspergillus fumigatus to less than 4% of that detected with AmB alone. In contrast, allicin did not enhance the cytotoxic activity of AmB against cells of human promyelocytic leukemia (HL-60), a vacuole-less organism. B1-35 Hereditary Spastic Paraplegia caused by Kinesin-1 mutants B. Ebbing 1 , K. Mann 1 , R. Schu ¨ le 2 and G. Woehlke 1 1 Institute for Cell Biology LMU, Munich, GERMANY, 2 Hertie-Institute for Clinical Brain Research, Tu ¨ bingen, GERMANY Hereditary Spastic Paraplegia is a neurodegenerative motor neuron disease characterized by lower limb spasticity and weakness. This autosomal dominant disease is linked to mutation of at least 20 genes, among them several point mutations in the neuronal Kinesin- 1 (KIF5A) gene. Here, for the first time, we investigate the in vitro properties of the wild-type KIF5A and three of the point mutations, which are located in the motor domain. The N256S mutant had a lower gliding velocity, whereas the R280S mutant exhibited low affinity to microtubules. The K253N mutant showed both a microtu- bule-affinity and a velocity defect. In laser trapping assays, none of the mutants moved more than a few steps on microtubules. Mixed gliding assays were utilized to show whether these defects can domin- ate the wild-type protein. Only the N256S mutant decreased the overall gliding velocity, when mixed one to one with the wild-type kinesin. Potentially only the N256S mutant can slow down the cargo transport in neurons. Assuming equal gene expression, patients are expected to have 25% wild type, 50%heterodimeric and 25% ho- modimeric kinesin. To simulate neuronal cargo transport we attached such mixtures to quantum dots and measured their veloci- ties. Our data indicate that a significant portion of cargo is transpor- ted at slower rates. B1-36 New cationic lipids as promising gene delivery carriers: effect of the structure on biological activity D. Medvedeva 1 , D. Rapoport 1 , A. Vladimirova 1 , N. Mironova 1 , M. Zenkova 1 , V. Vlassov 1 , M. Maslov 2 and G. Serebrennikova 2 1 Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, RUSSIAN FEDERATION, 2 Lomonosov Moscow State Academy of Fine Chemical Technology, Moskow, RUSSIAN FEDERATION Cationic lipids are extensively used as non-viral vectors for the gene transfer but a number of the drawbacks limit their use in vitro and in vivo. One of the ways to solve these problems is creating the new biodegradable non-toxic cationic lipids based on the natural compounds and studying their properties. We synthesized and tes- ted the new biodegradable cationic lipids based on the cholesterol and glycerol derivatives with the heterocyclic cationic head groups and various linkers and studied them as gene delivery carriers. It was found that the new cationic lipids are low-toxic at the concen- trations below 10 lM for the various cell lines (293, HeLa, BHK, BHK IR-780). By fluorescent microscopy and FACS we showed that the presence of the cationic lipids enhanced the delivery of various nucleic acids (oligonucleotides, plasmid DNA and siRNA) into cells. The transfection efficiency and toxicity of the new cati- onic lipids were compared with the commercially available cationic agent Lipofectamine. Hence, the obtained results suggest that the new cationic lipids based on the natural compounds could be the promising agents for the transfection of human cells. Acknowledgements: This work was supported by RAS pro- grams ‘‘Molecular and Cellular Biology’’ and ‘‘Science to Medi- cine’’, RFBR grant 05-04-48985. Transport Machineries Abstracts ª 2007 The Authors Journal compilation ª 2007 FEBS 105 B1-37 The LC8 family of dynein light chains: multifunctional chaperon-like proteins Z. Ho ´ di 1 , P. Rapali 1 , L. Radnai 1 , T. Molna ´ r 1 ,A ´ . Szenes 1 , J. Kardos 1 , L. Buday 2 , W. F. Stafford 3 and L. Nyitray 1 1 Eo ¨ tvo ¨ s Lora ´ nd University, Budapest, HUNGARY, 2 Semmelweis University, Budapest, HUNGARY, 3 Boston Biomedical Research Institute, Boston, MA, USA DYNLL1 and DYNLL2 are two mammalian paralogs of the con- served LC8 family of dynein light chains that were described as tail subunits of dynein and myosin Va motor proteins. Moreover, they were shown to interact with a wide variety of other polypeptides. They may serve as adaptors to bind cargos to the transport motors however, they also have other functions in the cell unrelated to their role in motor complexes. We have localized the binding site of DYN- LL2 on myosin Va tail within an uncoiled region, between two coiled-coil domains. DYNLL2 binds to this site with a Kd of 40 nM and stabilizes both flanking coiled-coils. By studying DYNLL bind- ing to their targets, we have found that PAK1, an important regula- tor of cell motility, regulates activity of DYNLL1, but not DYNLL2, by phosphorylating Ser88 and dissociating the homo- dimer protein into two monomers. The monomeric DYNLL (mim- icked by Ser88Glu mutation) is unable to bind to their known targets however, it may bind to yet undiscovered proteins. By analyzing the sequences of all known partners (>60), we have noticed that the only common feature of the DYNLL targets is that they are either intrin- sically disordered proteins (e.g. the proaptototic proteins Bim and Bmf) or have their DLC binding sites within a disordered domain (e.g. myosin Va and the dynein intermediate chain). Thus, DYNLL isoforms are now recognized as chaperon-like ‘‘hub proteins’’ with well-defined structure that could promiscuously bind to diverse inter- actors that contain a short binding motif within a disordered protein or domain and contribute to their folding and/or regulation. B1-38 Modified supramolecular concatemeric complexes as a novel system for oligonucleotide delivery into mammalian cells O. Gusachenko, D. Pyshnyi, M. Zenkova and V. Vlassov Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, RUSSIAN FEDERATION Application of nucleic acids for the inhibition of gene function by turning off genes with antisense oligonucleotides or double-stran- ded small interfering RNA has confidently gained recognition as a promising tool in the gene therapy. One of the main hurdles in the use of oligonucleotide-based therapeutics is their poor uptake by target cells. Recently formation of concatemeric complexes by oligonucleotides was shown to promote their binding to several mammalian cell lines [Simonova et al., 2006]. We proposed to use this phenomenon for the development of new oligonucleotides delivery system. To improve the efficiency of complexes penetration through the cellular membrane we attached lipophilic cholesterol molecules to different components of the concatemers. Uptake, cellular distribution and biological activity of the complexes formed by delivered oligonucleotides and cholesterol-modified carrier-oligo- nucleotides were studied. We show that incorporation of the desired antisense oligonucleotide into the self-assembling concatemeric sys- tem significantly promotes its delivery into cells without the addi- tion of any supplementary transfection agents and allows to achieve specific inhibition of the target gene. In contrast to the use of con- ventional delivery agents, concatemeric oligonucleotide system was shown to posses no cytotoxic and non-specific intercellular activit- ies. In conclusion, delivery of pharmacologically active oligonucleo- tides into the target cells in concatemeric form provides perspective alternative to the use of conventional transfection methods. Acknowledgements: This work was supported by RAS programs ‘‘Molecular and cellular biology’’ and ‘‘Science to Medicine’’. B1-39 The role of cytoplasmic networks in plasmid DNA intracellular trafficking and intranuclear movement of plasmid DNA V. Ondrej, E. Lukasova, S. Kozubek and M. Falk Institute of Biophysics Academy of Sciences of the Czech Republic, v.v.i., Brno, CZECH REPUBLIC The role of microtubule and actin networks in transport and pro- cessing of transfected plasmid DNA in human cells was studied. We observed strong binding activity of plasmid DNA to both networks, their immobilization at actin filaments and directional motion along the microtubules with average velocity v = 1.12 lm/min. In addition, actin filaments play an important role in dissociation of plasmid DNA aggregates at the cell periph- ery. Disruption of one of these networks led to the abortion of plasmid transport and accumulation of plasmid DNA in huge aggregates at the cell periphery. The microtubule network consti- tutes the high-ways for long-distance transport of plasmid DNA as a cargo towards the cell nucleus. Our results show also the functional role of actin in plasmid movement and targeting in the cell nucleus. The most frequent sites of plasmid targeting were those in which the genome integrity was impaired. Plasmid DNA frequently localized in double stranded DNA breaks (DSB) induced spontaneously or by ionizing radiation detected as sites with phosphorylated H2AX. Inhibition of actin polymerization by latrunculin B perturbed plasmid transport not only throughout cytoplasm but also inside the nucleus and resulted in stopping of plasmid co-localization with newly induced DSBs. B1-40 Cellular localization and trafficking of endogenous ATP7B and COMMD1 proteins S. Donadio, B. De Keukeleire, J. Micoud, F. Piccarreta and M. Benharouga CEA, Grenoble, FRANCE Copper (Cu), an essential trace element for cellular function, is highly cytotoxic when in excess. In the liver, a Cu-transporter ATPase (ATP7B) excretes the excess of Cu into the bile. A defect in ATP7B is associated with Wilson disease, a Cu-overload disor- der. Recently, COMMD1, a cytoplasmic protein responsible for liver’s Cu toxicosis, was shown to interact, in vitro, with the N- terminal region of ATP7B suggesting a cooperation for hepatic Cu detoxification. Today, the intracellular localization of ATP7B and COMMD1 is still a mater of controversy. In this study, we investigated in vivo interaction and endogenous localization of ATP7B and COMMD1 in human hepatoma cell line (HepG2). ATP7B and COMMD1 were co-immunoprecipitated in vivo inde- pendently of the cellular Cu content. Co-immunolocalization with intracellular compartment markers (endoplasmic reticulum, ER; Golgi apparatus, GA; early endosomes, EE; late endosomes/lyso- somes, LE/Ly and plasma membrane, PM) showed that 200 lM CuSO 4 stimulated trafficking of COMMD1 from ER, GA and EE to LE/Ly, as well as trafficking of ATP7B from ER and EE to the LE/Ly compartment. No GA and PM localization was observed. These results were sustained by those obtained after differential centrifugation and separation on sucrose gradient. Taken together these results suggested that the mechanism of bil- iary Cu excretion involves the intracellular trafficking of ATP7B/ COMMD1 and their interaction in the endosomes. Abstracts Transport Machineries 106 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... we analyzed the molecular identity of the transporters involved in the transport of vitamin C in isolated Sertoli cells and 42GPA9 Sertoli cell line WT1 was used as Sertoli cells specific marker to further validate the use of 42GPA9 cell line as a model system The kinetic assays revealed that both, ascorbic acid transporters (SVCTs) and facilitative hexose transporters (GLUTs), are functionally active... importance to the physiology of the germ cells (FONDECYT 1060135) 121 Abstracts Transport Machineries B4-18 Effect of specific inhibitors on pHi recovery of sertoli cells B4-20 A third nucleotide transporter (NTT) in Arabidopsis thaliana? P F Oliveira1, T Moura2, A Barros3, M Sousa4 and A R da Costa5,1 1 ´nico, CECA-UP, Porto, PORˆmetas e Transporte Io Lab Fisiol Ga TUGAL, 2CQFB, Department Quı´mica, FCT-UNL,... in heme utilization, the function exerted in conjunction with HmuR, an outer membrane heme transporter ª 2007 The Authors Journal compilation ª 2007 FEBS Transport Machineries Abstracts B4-22 CD and NMR studies of transmembrane segments of mitochondrial oxoglutarate carrier B4-24 MRS2 encodes the major Mg2+ transporter of human mitochondria F Bisaccia1, M A Castiglione Morelli1, A Ostuni1, F Armentano1... are grown on glucose, and an inducible active transport system, when cells are grown on phenanthrene Active transport followed Michaelis-Menten kinetics with a Kt value of 0.19 ± 0.06 lM and it was amenable to inhibition by 2,4-dinitrophenol and sodium azide Moreover, the saturation kinetics of the uptake implies specific binding of phenanthrene to the PAH-transporting system, with an apparent Kd of 0.48... (PDR), where overexpressed ABC transporters such as yeast PDR5 confer resistance to a vast variety of drugs PDR in yeast is similar to MDR occurring in mammalian tumor cells Little is known about physiological substrates for ABC transporters or their molecular mechanisms of function, mainly due to limited structural information Human ABC proteins have been implicated in the transport of lipids and sterols... hyperactive transcription factor Purified ABC transporters will be subjected to reconstitution experiments as well as structural studies to unravel their molecular mechanism of function, which could perhaps lead to a better understanding of clinically relevant ABC transporters B4-9 Localization and function of ABCC/MRP efflux pumps and organic anion uptake transporters in human brain A T Nies German Cancer... deficient mice This indicates that one of cellular nonapoAI:ABCA1 pathways must also highly contribute to cholesterol homeostasis in liver Involvement of ABCG1 transporter in this phenomenon will also be examined in future studies 120 Transport Machineries B4-12 Lateral compartmentation of proteins and lipids in the plasma membrane: involvement of the membrane potential M Opekarova, Sr.1, G Grossmann,... medium and the [3H] taurine uptake was reduced at the condition Also, we induced hyperglycemic condition using cytochalasin B, glucose transport inhibitor, and [3H] taurine uptake was reduced in TR-iBRB cells ª 2007 The Authors Journal compilation ª 2007 FEBS Transport Machineries B4-14 Cationic lipid-based nanostructures for gene delivery and imaging F Chang, C Chen, H Huang, K Chang, Y Lin, M Lin and... xanthine permease of E coli In this context, we analyzed 324QN325, a sequence motif conserved in all purine-transporting NATs, and the flanking 304DGLVSVIASAVGSLPLTTFA323 and 326NGVIQMTGVASRYVGR341 We found that: (a) Q324 is essential for high-affinity xanthine transport; (b) N325 is irreplaceable for active transport; (c) D304 is also essential, as mutants D304E, D304N and D304C display strikingly low uptake... of more than one single NLS and NES signal in the Methopren-tolerant, existing in different fragments of protein 107 Abstracts Transport Machineries B1-45 Characterisation of NLS in human PHD1 and PHD3 B2-2 The haemolysin A system of E.coli: from single subunits to the whole transport machinery A Hilz1,2, T Schillinger1, S G Schindler1, M Koehler2 and R Depping1 1 University of Luebeck, Luebeck, GERMANY, . of the rate of ER-to-Golgi transport in HEK293 cells, inhibition of cargo concentration of the EAAC1 glutamate transporter into transport complexes in HEK293. through the Golgi apparatus has a significant role in intra-Golgi transport. Abstracts Transport Machineries 102 ª 2007 The Authors Journal compilation ª 2007