Báo cáo khoa học: The b domain is required for Vps4p oligomerization into a functionally active ATPase potx

The b domain is required for Vps4p oligomerization into a functionally active ATPase Parimala R. Vajjhala 1 , Julin S. Wong 1 , Hui-Yi To 1 and Alan L. Munn 1,2,3,4 1 Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, Queensland, Australia 2 School of Biomedical Sciences, University of Queensland, St Lucia, Queensland, Australia 3 Laboratory of Yeast Cell Biology, Institute of Molecular and Cell Biology, A*STAR Biomedical Sciences Institutes, Singapore 4 Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore The multivesicular body (MVB) is a central sorting station in the itinerary of proteins that traffic through the endocytic pathway. In the MVB, integral mem- brane proteins that are destined for delivery to the lysosome lumen undergo MVB sorting into internal vesicles, which form by invagination of the limiting membrane of the MVB. This process sequesters the cytoplasmic tails of endocytosed signalling receptors and allows efficient silencing. Mature MVBs then fuse with the lysosome and transfer their contents, inclu- ding internal vesicles, into the lysosome lumen. In some cells, MVBs have been shown to fuse with the plasma membrane and have been suggested to function in intercellular signalling [1,2]. Moreover, the machin- ery that generates the internal vesicles of MVBs has recently been implicated in the budding of several clas- ses of enveloped viruses (reviewed in [3–5]). Both endocytic and biosynthetic traffic to the lyso- some proceed via the MVB [6,7], thus defects in the function of the MVB affect both endocytic transport Keywords class E vacuolar protein sorting; dopamine responsive gene-1; LYST-interacting protein 5; suppressor of K + uptake growth defect 1 (SKD1); SKD1-binding protein 1 Correspondence A. L. Munn, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD 4072, Australia Fax: +61 73346 2101 Tel: +61 73346 2017 E-mail: A.Munn@imb.uq.edu.au (Received 16 February 2006, revised 14 March 2006, accepted 20 March 2006) doi:10.1111/j.1742-4658.2006.05238.x Endocytic and biosynthetic trafficking pathways to the lysosome ⁄ vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome ⁄ vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endo- somal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a b domain, which protrudes from the AAA domain, and a final C-terminal a-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly con- served between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro.We show that the b domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic func- tions in vivo. We also show that the b domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function. Abbreviations CPY, carboxypeptidase Y; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; GST, glutathione S-transferase; MIT, microtubule interacting and trafficking; MVB, multivesicular body; PVDF, poly(vinylidene difluoride); Vps, vacuolar protein sorting. FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS 2357 and delivery of newly synthesized lysosomal ⁄ vacuolar proteins. Insights into both processes have come from studies of budding yeast vacuolar protein sorting (vps) mutants which missort soluble vacuolar proteins into the extracellular medium. A subclass of vps mutants, class E vps mutants, disrupt MVB sorting and form an enlarged multilamellar endosome adjacent to the vacu- ole termed the class E compartment [8–10]. This com- partment accumulates endocytic and biosynthetic material as well as recycling late Golgi proteins [9,11– 13] resulting from defective transport out of this aber- rant endosome. There are 27 class E Vps proteins in mammalian cells and 18 in yeast, which can be grouped into complexes referred to as endosomal sort- ing complexes required for transport (ESCRT I–III). These three complexes act sequentially to sort and deliver membrane proteins into forming intraluminal vesicles [14,15]. Vps4p, also known as Csc1p ⁄ End13p ⁄ Grd13p ⁄ Vpl4p ⁄ Vpt10p ⁄ Did6p, is a class E Vps protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family of ATPases [8,16]. Vps4p functions at multiple steps during endocytic transport [8,17] and has recently been shown to function in sterol metabolism [18]. There are two isoforms of VPS4 in human cells: VPS4A and VPS4B. The endocy- tic functions of yeast Vps4p are conserved in mamma- lian VPS4B [19–21], and both human VPS4A and VPS4B have been shown to be required for virus bud- ding [22,23]. Vps4p contains a single AAA domain, and its ATPase activity is critical for function as vps4 mutants defective in ATP binding (K179A) or hydroly- sis (E233Q), and a temperature-sensitive vps4 mutant (M307T ⁄ L327S) and the end13-1 mutant (S335F), which also have mutations in the AAA domain, all induce class E compartments and perturb endocytic and biosynthetic traffic [8,17]. Vps4p ATPase activity is important for disassembly of ESCRTs to allow reuse in multiple rounds of MVB sorting [24]. Whereas many AAA proteins form hexamers, wild- type Vps4p has not been shown to form a higher-order oligomer. Wild-type Vps4p purified from bacteria forms a dimer [24]. However, a homotypic interaction has not been demonstrated for wild-type Vps4p in vivo using a yeast two-hybrid assay [25]. Moreover, endo- genous human VPS4B appears to exist as a monomer [26]. Thus it is not clear whether Vps4p exists as a dimer in vivo or whether dimer formation is important for Vps4p function in vivo, and the structural determi- nants required for dimerization of Vps4p have not been identified. Although a homotypic interaction has been described for the Vps4p-E233Q mutant in a yeast two-hybrid assay [25], and this mutant forms a 10–12- mer in the presence of ATP [24,27], it is not clear whe- ther this is due to stabilization of a complex that is normally transiently formed by wild-type Vps4p or to formation of an aberrant complex. Consistent with the ability of Vps4p to form oligomers, most vps4 mutants that have been described to date are dominant negative [17,24,28]. The N-terminal region of Vps4p contains a microtu- bule interacting and trafficking (MIT) domain [29]. This region of Vps4p interacts with Vps20p ⁄ Chm6p [30], a component of ESCRT III, and is required to recruit Vps4p to endosomes. The C-terminal region of Vps4p binds to another class E Vps protein, Vta1p [30], and this interaction is conserved in mammalian cells [26]. As the C-terminal region of Vps4p contains several motifs that are highly conserved between yeast Vps4p and both of the human VPS4 isoforms, we hypothesized that this region has an important role in Vps4p assembly or function. In this study, we show that conserved motifs in the C-terminal region, which are mainly present in the recently identified b domain of Vps4p, are essential for interaction with Vta1p. In addition, we show that one of these motifs is required for a homotypic interaction and for formation of a highly active ATPase complex, but is not required for endosomal recruitment. More importantly, the con- served motifs in the b domain are required for Vps4p functions in vivo. Results Conserved motifs in the Vps4p C-terminal region are essential for function but not for protein stability The Vps4p sequence located C-terminal to the previ- ously predicted AAA domain [8] contains several motifs that are highly conserved in human VPS4B (Fig. 1A) as well as VPS4A (not shown). To examine the functional importance of these C-terminal motifs and the C-terminal region in general, we constructed various mutations in this domain and examined the effect of each on Vps4p stability and in vivo function. To assess the importance of the C-terminal region (res- idues 351–437) or half of it (residues 395–437), the corresponding C-terminal truncation mutants (Vps4p- Ter1 and Vps4p-Ter2, respectively) were generated. To assess the importance of individual conserved motifs, we chose three motifs to delete. These deletion mutant proteins are named according to the first three amino acids of each motif deleted, i.e. Vps4p-RKI, Vps4p- GAI and Vps4p-LTP. In addition, we generated a mutant in which each charged residue in a conserved Role of the Vps4 b domain P. R. Vajjhala et al. 2358 FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS motif (RDE) at the extreme C-terminus was substi- tuted with alanine (Vps4p-RDE). The recent crystal structure of human VPS4B [27] allowed us to map the positions of these motifs (Fig. 1A). The domain organization of yeast Vps4p (Fig. 1B) inferred from the elucidated 3D crystal struc- ture of human VPS4B shows that the AAA domain is unusual in that it contains a domain with three antipar- allel b-sheets, referred to as the b domain within the C-terminal subdomain. Therefore the AAA domain extends past the original predicted AAA domain. Thus Vps4p-Ter1 lacks the end of a-helix 8 in the AAA domain, the entire b domain and both of the two C-ter- minal a-helices. Vps4p-Ter2 lacks only the two C-ter- minal a-helices. The motif deleted in the Vps4p-RKI mutant is positioned at the end of a-helix 8 of the AAA domain and extends into the first b-sheet of the b domain. In comparison, the motifs deleted in the Vps4p- GAI and Vps4p-LTP mutants are positioned adjacent to and within the second b-sheet, and within the third b-sheet, respectively, of the b domain. The RDE motif subjected to charged-to-alanine substitutions in the Vps4p-RDE mutant is positioned at the end of the C-terminal a-helix (Fig. 1A). The positions of the highly conserved residues that were mutated are shown on the VPS4B structure in Fig. 1C. To test whether the Vps4p mutant proteins are sta- bly expressed in yeast, plasmids expressing the wild- type and mutant Vps4p constructs were introduced into vps4D cells, and Vps4p was detected in cell extracts by immunoblotting. The steady-state levels of Vps4p-RKI, Vps4p-GAI, Vps4p-LTP and Vps4p-RDE mutant proteins are comparable to wild-type Vps4p (Fig. 1D). In contrast, the level of Vps4p-Ter1 and Vps4p-Ter2 mutant proteins (38.3 and 43.9 kDa, respectively) was too low to detect (data not shown). Although the C-terminus itself may be required for protein stability, we conclude that the four conserved motifs that we mutated are not individually essential for protein stability. We next examined whether those Vps4p mutant con- structs that are stably expressed can substitute for wild- type Vps4p in its various functions. Plasmids expressing wild-type Vps4p or the various Vps4p mutant proteins or empty vector were introduced into vps4D yeast cells and the ability of the mutant proteins to rescue the A S.c. Vps4 351 IRKIQSATHFKDV STEDDE TRKLTPCSPGD 380 .::.::::::: : : : . :::::::: H.s. Vps4B 353 VRKVQSATHFKKVRGPSRADPNHLVDDLLTPCSPGD 388 S.c. Vps4 381 DGAIEMSWTDIEADELKEPDLTIKDFLKAIKSTRPT 416 :::::.: :. : : :: : : .:.:: H.s. Vps4B 389 PGAIEMTWMDVPGDKLLEPVVSMSDMLRSLSNTKPT 424 S.c. Vps4 417 VNEDDLLKQEQFTRDFGQEG437 ::: :::: :: :::::: H.s. Vps4B 425 VNEHDLLKLKKFTEDFGQEG 444 β6 β7 β8 α9 α10 α8 α10 C D empty vector Vps4p-GAI Vps4p-LTP Vps4p-RKI Vps4p-RDE Vps4p actin Vps4p B AAA N-terminal subdomain AAA C-terminal subdomain β domain C-terminal helix MIT domain 1 437 299 129 358 399 415 79 Fig. 1. Construction of Vps4p C-terminal mutants. (A) Alignment of S. cerevisiae (S.c) Vps4p and human (H.s) VPS4B sequences using LALIGN [50]. Conserved blocks deleted in individual mutants are shown in bold, and residues that were substituted with alanine in the RDE mutant are shown in bold italics. The secondary structure of the corresponding region of VPS4B is also shown. (B) Schematic representation of wild-type Vps4p with the domain organization inferred from structural data of VPS4A and VPS4B [27,34]. (C) Locat- ion of highly conserved residues in the human VPS4B structure that were mutated in yeast Vps4p. The b domain is circled, and the RKI, LTP and GAI motifs are shown in red, light blue and green, respectively. The charged residues in the RDE motif are shown in dark blue. The colour code for the nonmutated residues in the dif- ferent domains is: b domain, orange; N-terminal AAA subdomain, pink; C-terminal AAA subdomain, light brown; C-terminal a-helix, brown. (D) Total cell lysates from RH2906 (vps4D) yeast cells carry- ing plasmids expressing wild-type Vps4p, Vps4p-RDE, Vps4p-RKI, Vps4p-LTP and Vps4p-GAI mutant proteins or carrying empty vector (YCplac111) were subjected to western blotting using either a poly- clonal antibody to Vps4p or a monoclonal antibody to actin. P. R. Vajjhala et al. Role of the Vps4 b domain FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS 2359 phenotypes of vps4D cells was assessed. Vacuolar accu- mulation of a fluid-phase endocytic marker, MVB sort- ing of a membrane protein to the vacuole lumen, delivery of soluble vacuolar proteins to the vacuole and growth at high temperature were examined. Endocytosis and subsequent vacuolar accumulation of the fluid-phase marker, Lucifer Yellow, was restored by wild-type Vps4p but not by Vps4p-RKI, Vps4p- GAI or Vps4p-LTP mutant forms of Vps4p when compared with empty vector alone (Fig. 2A). In con- trast, vacuolar accumulation of Lucifer Yellow was efficiently restored by Vps4p-RDE. To examine MVB sorting, we used the iron transporter homologue, Fth1p, fused to ubiquitin (Fth1p-Ub) as a marker. Fth1p normally resides on the limiting membrane of the vacuole, but, when tagged with ubiquitin, it under- goes ubiquitin-dependent MVB sorting and is delivered to the vacuole lumen [31]. Green fluorescent protein (GFP)-tagged Fth1p-Ub was mainly observed in a class E compartment adjacent to the vacuole in vps4D cells expressing Vps4p-RKI, Vps4p-GAI or Vps4p- LTP similar to vps4D cells carrying the empty vector (Fig. 2B). The small amount that reached the vacuole was present on the limiting membrane. In contrast, expression of wild-type Vps4p or Vps4p-RDE resulted in delivery of GFP-tagged Fth1p-Ub to the vacuole lumen. These data clearly demonstrate that the con- served C-terminal motifs that are required for efficient fluid-phase endocytosis are also critical for ubiquitin- dependent MVB sorting. To assess the ability of the mutant Vps4p con- structs to function in vacuolar protein sorting, we assayed their ability to correct the missorting and secretion of carboxypeptidase Y (CPY) in vps4D cells. CPY is a soluble resident vacuolar protein that is translocated into the endoplasmic reticulum and then transported to the Golgi where a receptor, Vps10p, sorts it from secretory proteins destined for the cell surface into a pathway that takes it via endosomes to the vacuole. In vps4D cells, CPY is missorted at the late Golgi into vesicles destined for the cell surface and secreted [8]. Expression of wild- type Vps4p but not Vps4p-RKI, Vps4p-GAI or Vps4p-LTP mutant proteins restored vacuolar deliv- ery of CPY compared with empty vector alone (Fig. 2C). In contrast, CPY sorting to the vacuole is restored by Vps4p-RDE. Finally, we examined the ability of the various mutant Vps4p constructs to function in cell growth at high temperature. vps4D cells expressing wild-type or mutant Vps4p proteins or carrying empty vector were tested for growth on solid medium at high tempera- ture. Expression of wild-type Vps4p but not Vps4p-RKI, Vps4p-GAI and Vps4p-LTP or empty vector rescued growth at 40 °C (Fig. 2D). In contrast, Vps4p-RDE was able to significantly restore growth at 40 °C. However, Vps4p-RDE was reproducibly less efficient than wild-type Vps4p in restoring growth at 40 °Ctovps4D cells. There was no obvious difference in the growth rate at 24 °C between vps4D cells expres- sing wild-type Vps4p, any of the Vps4p mutants or those carrying empty vector. We conclude that the conserved motifs adjacent to and within the b domain that were deleted in the Vps4p-RKI, Vps4p-GAI and Vps4p-LTP mutants are essential for all Vps4p functions tested but not for pro- tein stability. The charged residues in the RDE motif at the end of the C-terminal helix of Vps4p are not essen- tial for protein stability or for most functions, however, they are required for full cellular growth at 40 °C. Fig. 2. Conserved motifs within and adjacent to the b domain of Vps4p are essential for several Vps4p in vivo functions. (A) Lucifer Yellow uptake and accumulation in the vacuole was measured in RH2906 (vps4D) yeast cells carrying plasmids expressing wild-type Vps4p or Vps4p mutant proteins or carrying empty vector (YCplac111). The same fields of cells are shown visualized by fluorescence (left) and Nomar- ski (right) optics. The vacuoles appear as indentations in the cell profile by Nomarski optics. Scale bar, 5 lm. (B) Ubiquitin-dependent MVB sorting of Fth1p-GFP-Ub in AMY245 (vps4D) yeast cells carrying plasmids expressing wild-type Vps4p or Vps4p mutant proteins or carrying empty vector (YCplac111). Cells were incubated in YPUAD medium containing 100 l M bathophenanthroline disulfonic acid for 6 h to chelate iron and induce Fth1p-GFP-Ub expression. Cells were then washed with buffer containing 1% sodium azide, 1% sodium fluoride, 100 m M phosphate, pH 8.0, to stop further transport. The same fields of cells are shown visualized by fluorescence (left) and Nomarski (right) optics. Scale bar, 5 lm. (C) Vacuolar protein sorting in RH2906 (vps4D) yeast cells carrying plasmids expressing wild-type Vps4p or Vps4p mutant proteins or carrying empty vector (YCplac111) or no vector. Cells were grown on YPUAD solid medium for 2 days at 24 °C in contact with a nitrocellulose filter. RH1800 (wild-type) yeast cells without any vector (boxed in both panels) was included as a control. Cells were eluted from the filter, and CPY on the filter was detected by immunoblotting with anti-CPY serum. To test for cell lysis, the blot was stripped and reprobed with an antibody to a cytoplasmic protein (calmodulin). (D) Temperature-sensitive growth assay of RH2906 (vps4D) yeast cells carrying plasmids expressing wild-type Vps4p or Vps4p mutant proteins or carrying empty vector (YCplac111). Cells were serially diluted 10-fold, and 7-lL aliquots were spotted on to YPUAD solid medium and incubated at 24 °C (left) or 40 °C (right). Plates were photographed after 4 or 12 days, respectively. Role of the Vps4 b domain P. R. Vajjhala et al. 2360 FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS Conserved motifs adjacent to and within the Vps4p b domain are required for interaction with Vta1p, but not Vps20p or Did2p A number of Vps4p-interacting proteins have previ- ously been identified in a yeast two-hybrid screen including the class E Vps proteins Vps20p and Vta1p [30]. These interactions were confirmed by the demon- stration that recombinant glutathione S-transferase (GST)-Vps20p or GST-Vta1p bind GFP-tagged Vps4p present in yeast cell lysates as well as to recombinant His 6 -tagged Vps4p in vitro. Using yeast Vps4p-GAI Vps4p-L TP Vps4p-RDE Vps4p-WT empt y vector Vps4p-RKI Fluorescence Nomarski Vps4p-GAI Vps4p-LTP Vps4p-RDE Vps4p-WT empty vector Vps4p-RKI Fluorescence Nomarski A B C Vps4p-WT Vps4p-GAI Vps4p-RDE no vector empty vector Vps4p-RK I Vps4p- LT P wild-type Vps4p-WT Vps4p-GAI Vps4p-RDE no vector empty vector Vps4p-RK I Vps4p- LT P wild-type CPY blo t Calmodulin blo t D Vps4p-WT empty vector Vps4p-RK I Vps4p-GAI Vps4p-L TP Vps4p-RDE 40 C O 24 C O P. R. Vajjhala et al. Role of the Vps4 b domain FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS 2361 two-hybrid assays, Vps20p was shown to interact strongly with the N-terminal region of Vps4p and weakly with both full-length Vps4p and a C-terminal region containing residues 351–437. Vta1p interacts strongly with full-length Vps4p and with the C-ter- minal region, but does not interact with the N-ter- minal region. Did2p, also known as Chm1p ⁄ Fti1p ⁄ Vps46p, is also a class E Vps protein which interacts with Vps4p [30,32], and this interaction is also conserved in mammalian cells [33]. We used a yeast two-hybrid assay to determine the region of Vps4p that interacts with Did2p (Fig. 3A). Did2p binds strongly to full-length Vps4p and, like Vps20p, binds very strongly to the N-terminal region of Vps4p and only weakly to the C-terminal region. This is consistent with a recent finding that the MIT domain of VPS4A interacts with CHMP1B [34]. Loss of interaction with these proteins may be responsible for loss of Vps4p function caused by the C-terminal mutations. We therefore tested whether the conserved motifs in the C-terminal region of Vps4p are required for two-hybrid interaction with each of these proteins (Fig. 3B). Vta1p interacted with wild-type Vps4p but not with the Vps4p-RKI, Vps4p-GAI or Vps4p-LTP mutant proteins. In contrast with the other mutant proteins, Vps4p-RDE retained the ability to interact with Vta1p. As expected, all the Vps4p mutant proteins retained the ability to interact with Vps20p. This interaction, which is mediated by the N-terminal region, should not be directly affected by C-terminal mutations. Interestingly Vps4p-RKI, Vps4p-GAI and Vps4p-LTP exhibited an apparent increase in interac- tion with Vps20p. The interaction with Did2p was unaffected by any of the mutations. A Vps4p pLexA Vps4p-RDE Vps4p-RKI Vps4p-GAI Vps4p-LTP pB42AD Did2p Vps20p Vta1p B pB42AD Did2p Vps20p Vta1p pLexA Vps4p N-Vps4p C-Vps4p AAA-Vps4p C GST-Vps20p GST-Vta1p GST ATP + - + ++ - bound unbound bound unbound bound unbound +-+-ATP ATP Vps4p-6His Vps4p-GAI-6His Vps4p-6His Vps4p-GAI-6His Vps4p-6His Vps4p-GAI-6His Fig. 3. Role of conserved Vps4p C-terminal motifs in interaction with Vta1p, Vps20p and Did2p. (A) Yeast two-hybrid interaction anal- ysis of Did2p (residues 41–204 ⁄ end) with full-length wild-type Vps4p (residues 1–437 ⁄ end), the N-terminal region of Vps4p (N-Vps4p; residues 1–128), the previously predicted AAA domain (AAA-Vps4p; residues 129–350) [8] and C-terminal region (C-Vps4p; residues 351–437 ⁄ end). The interaction analyses with Vta1p (residues 108–333 ⁄ end) and Vps20p (residues 3–221 ⁄ end) were included as controls. (B) Yeast two-hybrid interaction analysis of wild-type Vps4p and Vps4p C-terminal mutants with the same frag- ments of Vta1p, Vps20p and Did2p as in (A). In (A) and (B), EGY48 carrying pLexA-based bait plasmids and pB42AD-based prey plas- mids as well as p8op-LacZ reporter plasmid were spotted on to medium containing X-gal. Plates were photographed after overnight incubation, and two-hybrid interaction was assessed by blue color- ation. Two independent transformants are shown in (B). (C) In vitro binding of His 6 -tagged wild-type Vps4p and Vps4p-GAI to GST- Vps20p and GST-Vta1p. Equal amounts of full-length His 6 -tagged proteins were incubated with glutathione ⁄ agarose bearing GST- Vta1p, GST-Vps20p or GST alone in the presence or absence of ATP. The unbound protein was recovered in the supernatants. Bound protein was released with Laemmli sample buffer. The bound and unbound fractions were subjected to SDS ⁄ PAGE and immunoblotting with a polyclonal antibody to Vps4p. The amount of wild-type Vps4p-His 6 or Vps4p-GAI-His 6 bound to GST-Vps20 was quantified by densitometry. The shift in the relative positions of the wild-type Vps4p-His 6 and Vps4p-GAI-His 6 bound to GST-Vps20 is due to the presence of the GST-Vps20 protein, which migrates very close to the His 6 -tagged proteins. Role of the Vps4 b domain P. R. Vajjhala et al. 2362 FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS Because yeast two-hybrid is an indirect measure of binding strength, we used in vitro protein-binding assays to confirm the effect of the Vps4p-GAI muta- tion on the interaction with Vps20p and Vta1p. Both wild-type Vps4p and Vps4p-GAI were expressed with His 6 affinity tags in Escherichia coli and used in binding assays with GST-Vta1p and GST-Vps20p (Fig. 3C). Consistent with the yeast two-hybrid data, the Vps4p-GAI-His 6 mutant protein did not interact with Vta1p. Also consistent with our yeast two-hybrid results, binding to GST-Vps20p was increased com- pared with wild-type Vps4p-His 6 (both in the presence or absence of ATP). In the presence of ATP, there was a 35% decrease in the amount of Vps4p-GAI bound to GST-Vps20p. In contrast, there was an 87% decrease in the amount of wild-type Vps4p bound to GST-Vps20p. Hence, the ATPase-dependent dissoci- ation of GST-Vps20p is affected by deletion of the conserved GAI motif in Vps4p. We conclude that the RKI, GAI and LTP motifs within and adjacent to the Vps4p b domain are essential for interaction with Vta1p. The increased interaction with Vps20p, when these motifs are deleted may be due to defective ATPase-depend- ent dissociation, which we showed directly for Vps4p-GAI. The Vps4p GAI motif in the b domain is not required for Vps4p recruitment to endosomes The Vps4p N-terminal domain has been shown to play a key role in recruitment of Vps4p to endosomes, but whether recruitment also requires the C-terminal domain was not tested [24]. To test whether the C-ter- minal GAI motif is important for Vps4p recruitment to endosomes, we examined the subcellular localization of GFP-tagged Vps4p-GAI and compared it with that of GFP-tagged wild-type Vps4p (Fig. 4A). In vps4D yeast cells, the fluorescence distribution of wild-type Vps4p-GFP and Vps4p-GAI was both diffuse and localized to punctate structures that are likely to be endosomes. In contrast, the subcellular distribution of GFP-tagged Vps4p-DCC, which lacks the N-terminal MIT domain, was diffuse in the cytoplasm. This is consistent with a critical role for the N-terminal MIT domain in recruitment of Vps4p to endosomal mem- branes that has previously been described [24]. Fur- thermore, when the DCC mutation was introduced into Vps4p-GAI, the subcellular distribution also became diffuse in the cytoplasm. We conclude that Vps4p-GAI, but not Vps4p-DCC-GAI, is recruited to endosomal membranes. This suggests that the C-ter- minal GAI motif is not essential for Vps4p recruitment to endosomes. The Vps4p GAI motif is not essential for ATPase activity To determine whether the GAI motif in the b domain is important for Vps4p ATPase activity, perhaps via con- formational effects on the AAA domain, we assayed the ATPase activity of His 6 -tagged Vps4p-GAI and com- pared it with that of His 6 -tagged wild-type Vps4p. The affinity-purified Vps4p-GAI-His 6 mutant protein was difficult to obtain as a full-length protein from bacteria and most preparations contained some degradation products. In the best preparations,  30% of the protein was full-length as determined by densitometry. When equivalent amounts of full-length protein were assayed in the presence of 0.1 mm ATP, the Vps4p-GAI-His 6 protein had 52% of wild-type activity (Fig. 5). However, when equal amounts of full-length protein were assayed in the presence of 1 mm ATP, the Vps4p-GAI-His 6 pro- tein had only 14% of wild-type activity. We conclude that the Vps4p GAI motif in the b domain is important, although not essential for Vps4p ATPase activity. Fluorescence Nomarski vps4∆/ Vps4p-GFP vps4∆/ Vps4p-GAI-GFP vps4∆/ Vps4p-∆CC-GFP vps4∆/ Vps4p-∆CC-GAI-GFP Fig. 4. The GAI motif in the b domain is not essential for localiz- ation of Vps4p to endosomes. (A) RH2906 vps4D yeast cells expressing GFP-tagged wild-type Vps4p, Vps4p-GAI, Vps4p-DCC or Vps4p-DCC-GAI were grown in SD medium, and the GFP-tagged proteins visualized by fluorescence microscopy. Scale bar, 5 lm. P. R. Vajjhala et al. Role of the Vps4 b domain FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS 2363 The phenotypes conferred by mutation of the conserved motifs adjacent to and within the Vps4p b domain are not dominant To investigate whether mutation of the Vps4p RKI, GAI and LTP motifs confers dominant phenotypes, like most previously characterized vps4 mutants, we expressed the mutant proteins in wild-type cells and examined the effect on Vps4p function. Wild-type cells expressing the Vps4p-RKI, Vps4p-GAI and Vps4p-LTP mutants did not missort and secrete CPY, grew well at 40 °C, and transported Fth1p- Ub-GFP to the vacuole lumen (data not shown). In contrast, wild-type cells expressing the dominant-neg- ative Vps4p-E233Q mutant [24] showed the full range of dominant-negative phenotypes. This indi- cates that the defects conferred by mutation of the Vps4p-RKI, Vps4p-GAI and Vps4p-LTP motifs are not dominant. An intact b domain is essential for Vps4p self-association Although wild-type Vps4p expressed in bacteria is a dimer [24], previous studies using the yeast two-hybrid technique did not reveal a homotypic interaction in wild-type Vps4p [25]. We used a different yeast two- hybrid system to determine whether we could detect this homotypic interaction. The data obtained show that wild-type Vps4p can interact with itself strongly (Fig. 6). This is consistent with previous data showing that Vps4p forms a dimer. As the b-domain mutants are recessive, we consid- ered the possibility that these mutants may not be able to interact with wild-type Vps4p. To test this, we used the yeast two-hybrid assay to study the interaction between Vps4p-GAI and wild-type Vps4p. However, the Vps4p-GAI mutant protein barely interacted with wild-type Vps4p and did not interact with itself (Fig. 6). The Vps4p-RKI and Vps4p-LTP mutant pro- teins also did not interact with wild-type Vps4p (data not shown). We surmise that an intact b domain is required for Vps4p self-association. An intact b domain is required for the Vps4p-E233Q mutant to have a dominant- negative phenotype The E233Q mutation in Vps4p confers a dominant- negative phenotype [8,28], and the equivalent muta- tion in mammalian VPS4 isoforms has been widely used to study the effect of VPS4 inactivation in mammalian cells [35,19]. Vps4p-E233Q forms a 10– 12-mer [24,27], and it has been speculated that wild- type Vps4p may also form a high-molecular-mass oligomer that is transient in the presence of a func- tional ATPase domain. The dominant-negative pheno- type conferred by Vps4p-E233Q is believed to be due to interaction of Vps4p-E233Q with wild-type Vps4p in vivo based on the ability of Vps4p-E233Q to oligo- 1.0 0.1 GAI WT GAI WT [ATP] m M 0 5 10 15 20 25 nmol inorganic phosphate released /h/µg full-length protein Fig. 5. The Vps4p-GAI mutant has a diminished ATPase activity. Affinity-purified His 6 -tagged wild-type Vps4p (WT) and Vps4p-GAI (GAI) were assayed in vitro for ATPase activity at 30 °C. The amount of protein assayed was normalized based on the level of full-length wild-type Vps4p and Vps4p-GAI. ATPase activity was assayed in the presence of 0.1 m M ATP or 1 mM ATP and is expressed as nmol inorganic phosphate releasedÆh )1 Æ(lg full-length protein) )1 and shown graphically. pLexA/ pB42AD pLexA Vps4p/ pB42AD Vps4p pLexA Vps4p/ pB42AD Vps4p-GAI pLexA Vps4p-GAI/ pB42AD Vps4p pLexA Vps4p-GAI/ pB42AD Vps4p-GAI Fig. 6. The conserved GAI motif in the b domain of Vps4p is required for homotypic interaction between wild-type Vps4p mole- cules. The interaction between various combinations of wild-type Vps4p and Vps4p-GAI was assessed using the yeast two-hybrid technique. EGY48 carrying a p8op reporter plasmid and pLexA- based bait plasmids and pB42AD-based prey plasmids were spot- ted on to synthetic galactose medium containing X-gal. Plates were photographed after 2 days, and two-hybrid interaction was assessed by blue coloration. Three independent transformants are shown. Role of the Vps4 b domain P. R. Vajjhala et al. 2364 FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS merize with Vps4p-DCC [24] in vitro. To demonstrate a role for the b domain in Vps4p assembly in vivo, we mutated the GAI motif in the dominant-negative Vps4p-E233Q mutant. Unlike Vps4p-E233Q, the resulting double mutant (Vps4p-E233Q-GAI) did not confer dominant-negative phenotypes (Fig. 7A–C). As the expression level of Vps4p-E233Q-GAI is similar to that of Vps4p-E233Q (Fig. 7D), loss of the domin- ant-negative phenotype is not due to decreased expression of the double mutant. Our data strongly suggest that the b domain is required for interaction with wild-type Vps4p in vivo. Vps20p and Vta1p both stimulate Vps4p ATPase activity, but Vps20p stimulates its activity to a greater extent As the b domain of Vps4p is required for full ATPase activity as well as for interaction with Vta1p, it is poss- ible that binding of Vta1p to Vps4p might have an effect on ATPase activity. To test this hypothesis, we assayed the in vitro ATPase activity of wild-type His 6 - tagged Vps4p in the presence of increasing concentra- tions of GST-Vta1p. We also assayed the activity of Vps4p in the presence of increasing concentrations of GST and GST-Vps20p. We included GST-Vps20p because Vps20p binds to Vps4p in an ATP-sensitive manner, and substrate binding is known to increase the activity of some AAA ATPases such as Hsp104 and Katanin [36,37]. The data in Fig. 8 show that both Vta1p and Vps20p have a stimulatory effect on Vps4p ATPase activity, but the effect of Vps20p is much greater. It is interesting that, although the b domain is required for assembly of Vps4p into a complex with full ATPase activity, binding of Vta1p to the b domain does not inhibit Vps4p ATPase activity. Discussion Here we identify conserved motifs in the C-terminal region of Vps4p and provide evidence that those within the b domain are critical for all Vps4p in vivo A D Vps4p-E233Q Vps4p-E233Q-GAI Vps4p actin Vps4p- E233Q Vps4p- E233Q- GAI Fluorescence Nomarski C 24 O C 40 O C Vps4p-E233Q Vps4p-E233Q-GAI B Vps4p-E233Q Vps4p-E233Q-GAI Vps4p-E233Q Vps4p-E233Q-GAI CPY blot calmodulin blot Fig. 7. The dominant-negative Vps4p-E233Q mutant becomes recessive upon mutation of the GAI motif. (A) The Vps4p-E233Q-GAI mutant protein does not confer a dominant-negative MVB sorting defect. RH1800 (wild-type) yeast cells expressing Fth1p-GFP-Ub and either Vps4p- E233Q or Vps4p-E233Q-GAI were grown in SD selective medium and assayed for MVB sorting as in Fig. 2. Scale bar, 5 lm. (B) The Vps4p- E233Q-GAI mutant protein does not confer a dominant-negative vacuolar protein sorting defect. RH1800 (wild-type) yeast cells expressing Vps4p-E233Q or Vps4p-E233Q-GAI were grown on selective SD solid medium at 24 °C in contact with a nitrocellulose filter. CPY missorting was assayed as in Fig. 2. (C) The Vps4p-E233Q-GAI mutant protein does not confer a dominant-negative growth defect. Wild-type RH1800 yeast cells expressing Vps4p-E233Q or Vps4p-E233Q-GAI were assayed for growth at high temperature on solid SD selective medium as in Fig. 2. (D) Total cell lysates from RH2906 (vps4D) yeast cells carrying plasmids expressing Vps4p-E233Q or Vps4p-E233Q-GAI were subjec- ted to western blotting as in Fig. 1D. P. R. Vajjhala et al. Role of the Vps4 b domain FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS 2365 functions including fluid-phase endocytosis, MVB sort- ing, vacuolar protein sorting and growth at high tem- perature. Two of these motifs, LTP and GAI, are in the b domain and the third, RKI, is partly within the b domain and partly within the AAA domain. We pro- vide evidence that the b domain is important for full ATPase activity of Vps4p. We also show that the b domain is required for two protein interactions. The first is a homotypic interaction that may be important for assembly of a fully catalytically active oligomer, and the second is with Vta1p. Both of these interac- tions are likely to be important for Vps4p function in vivo. We also show that the charged residues in an RDE motif at the end of the final C-terminal a-helix are not required for most Vps4p functions but are required for full growth at high temperature. More- over, these charged residues are not required for Vta1p interaction. Several lines of evidence suggest that mutation of these motifs have specific effects on Vps4p in vivo func- tion. Deletion of the motifs did not compromise stable expression, indicating that loss of in vivo function is not merely due to lowered expression levels. The Vps4p proteins carrying mutations in the b domain retained at least part of their native structure, as they were able to interact with Vps20p and Did2p, which interact with the N-terminal region of Vps4p ([30]; this study). This was shown by yeast two-hybrid assay and confirmed in one b domain mutant using an in vitro binding assay. In addition, the retention of native structure in b domain mutant proteins is supported by the ability of a b domain mutant protein to be recrui- ted efficiently to endosomes in vivo as assessed visually by microscopy. Hence it is possible to mutate the b domain without grossly affecting Vps4p structure. Moreover, based on the structure of the human VPS4B monomer, the b domain is an independent domain that is separated from the AAA domain by random coils (Fig. 1E), thus mutations in this domain are unli- kely to perturb the structure of the AAA domain. However, we cannot exclude the possibility that dele- tion of the conserved motifs within the b domain may perturb the local structure of the b domain. The requirement for an intact b domain for full ATPase activity is clearly demonstrated in the in vitro ATPase assays and further supported by interaction analysis of the b domain mutant with Vps20p. The Vps4p–Vps20p interaction is known to be sensitive to ATP hydrolysis and is stabilized by the E233Q muta- tion that perturbs Vps4p ATPase activity [30]. Consis- tent with a decreased ATPase activity, interaction of the Vps4p b domain mutant with Vps20p was enhanced in the in vitro binding assay and in the in vivo yeast two-hybrid assay. Most AAA ATPases assemble into higher-order oligomeric rings. Consistent with this, wild-type Vps4p purified from bacteria forms a dimer [24], and Vps4p forms a 10–12-mer when its ATPase activity is compromised and it is locked in the ATP-bound conformation, as in the Vps4p-E233Q mutant [24,27]. Arg352 in the AAA domain of Vps4p has recently been shown to be important for assembly of Vps4p-E233Q into oligomers but not for dimer for- mation [27]. The motifs of Vps4p required for dime- rization have not previously been identified. Previous studies using a yeast two-hybrid assay have demon- 10 23456789 0 2 4 6 8 ratio of GST alone or GST-fusion protein (µg): Vps4p (µg) nmol inorganic phosphate released /h/µg Vps4p GST alone+Vps4p GST alone GST-Vps20p+Vps4p GST-Vps20p GST-Vta1p+Vps4p GST-Vta1p Fig. 8. Vps20p strongly stimulates Vps4p ATPase activity, but binding of Vta1p to the b domain of Vps4p has only a marginal stimulatory effect. The ATPase activity of recombinant Vps4p-His 6 was assayed at 30 °C in the presence of 0.3 mM ATP and increasing amounts of recombinant GST-Vta1p, GST-Vps20p or GST alone. ATPase activity is expressed as nmol inorganic phosphate releasedÆh )1 Æ(lg Vps4p) )1 . The ATPase activities of GST alone and the GST fusion proteins without any Vps4p were also assayed and are shown. Role of the Vps4 b domain P. R. Vajjhala et al. 2366 FEBS Journal 273 (2006) 2357–2373 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... 5¢-GCCCATATTCGTCGACGCGCTAACAGGTACCAGAGGAGAAGGAGAGAGCGAAGCAAGTAG-3¢ 5¢-GGGCGGATCCTCTGCTTTTCTTTATC-3¢ 5¢-GCGCTAATGCAACCGTAGTCAATTGATTAACGTGCT-3¢ 5¢-AGCACGTTAATCAATTGACTACGGTTGCATTAGCGC-3¢ 5¢-TTAAAAGAACCAGATTAGTCAATTGATTAACGTGCT-3¢ 5¢-AGCACGTTAATCAATTGACTAATCTGGTTCTTTTAA-3¢ 5¢-AAGCAAGAACAGTTCACTGCAGCTTTTGGTCAAGCAGGTAACTAGTCAATTGAT-3¢ 5¢-ATCAATTGACTAGTTACCTGCTTGACCAAAAGCTGCAGTGAACTGTTCTTGCTT-3¢ 5¢-GCGCTAATGCAACCGATAGATGTCTCTACGGAGGAC-3¢... oligomerization stimulates ATPase activity in a number of AAA and AAA+ ATPases including RuvB and FtsH [41,42] This is achieved by allosteric stimulation of ATPase activity by one or two conserved arginine residues in the < /b> N-terminal subdomain of the < /b> AAA+ or AAA domains (respectively) that contact the < /b> c-phosphate of ATP bound to an adjacent subunit The < /b> role of the < /b> AAA+ domain in oligomerization of AAA+... 5¢-GCGCTAATGCAACCGATAGATGTCTCTACGGAGGAC-3¢ 5¢-GTCCTCCGTAGAGACATCTATCGGTTGCATTAGCGC-3¢ 5¢-GACGACGAAACAAGAAAAGATGGCGCCATCGAGATG-3¢ 5¢-CATCTCGATGGCGCCATCTTTTCTTGTTTCGTCGTC-3¢ 5¢-TGCTCTCCAGGTGATGATATTGAAGCTGATGAATTA-3¢ 5¢-TAATTCATCAGCTTCAATATCATCACCTGGAGAGCA-3¢ Upstr F SalI R SalI F Dstr R Ter1 F Ter1 R Ter2 F Ter2 R RDE F RDE R RKI F RKI R LTP F LTP R GAI F GAI R To generate full-length VPS4, 5¢ and 3¢ PCR... Vps4 SalI F and Vps4 Dstr R primers Table 1 Yeast strains used in this study Strain Genotype Source Y15588 RH1201 MATa vps4-D::KanMx his3 leu2 lys2 ura3 MATa ⁄ MATa his4 ⁄ his4 leu2 ⁄ leu2 ura3 ⁄ ura3 lys2 ⁄ lys2 bar1 ⁄ bar1 MATa his4 leu2 ura3 bar1 MATa vps4-D::URA3 his4 leu2 ura3 lys2 bar1 MATa vps20-D::KanMx his4 leu2 ura3 bar1 MATa vps4-D::KanMx leu2 ura3 his4 lys2 bar1 EUROSCARF Riezman lab strain... Journal compilation ª 2006 FEBS 2367 Role of the < /b> Vps4 b domain P R Vajjhala et al hexamerization of the < /b> N-terminal AAA domain [43] Furthermore, Katanin requires microtubule binding for oligomeric assembly [44] As AAA+ proteins are studied, more examples of such proteins that require regions outside the < /b> AAA+ domain for assembly may become apparent The < /b> function of the < /b> C-terminal a- helix of Vps4p has not been... effects of Vps4pE233Q, further suggesting that the < /b> b domain is required for association with wild-type Vps4p in vivo Thirdly, in the < /b> presence of an increased concentration of ATP, we observed a substantial increase in the < /b> ATPase activity of wild-type Vps4p However, the < /b> increase in activity of a b- domain mutant was not as dramatic, suggesting that this mutant cannot assemble into a fully active ATPase Our... of AAA+ proteins is well established Our study, however, shows that for Vps4p, regions outside of the < /b> AAA domain are also required for assembly Although this finding was unexpected, there is a precedent for the < /b> role of regions outside of the < /b> AAA domain in oligomerization For example, the < /b> linker region between the < /b> two AAA domains of p97 is required for FEBS Journal 273 (2006) 2357–2373 ª 2006 The < /b> Authors... mutation of Arg352 in the < /b> RKI motif abolished interaction with Vta1p This mutation (R35 2A) is in the < /b> AAA domain and also prevents oligomerization of Vps4p dimers into a higher-order oligomer In addition, mutations in Role of the < /b> Vps4 b domain the < /b> RKI and LTP motifs (D36 2A and S37 7A, respectively) that are within the < /b> b domain also abolished the < /b> Vta1p interaction However, the < /b> effect of these b domain mutants... may be only partially functional endocytosis at high temperature In summary, the < /b> b domain of Vps4p is not required for recruitment to endosomes It appears to play a critical role in the < /b> formation of a Vps4p ATPase complex that is functional in vivo The < /b> formation of the < /b> latter may be a prerequisite for interaction with Vta1p Furthermore, our data highlight the < /b> importance of Vps4p assembly during MVB... AMY174 AMY245 2368 Riezman lab strain [17] [30] This study FEBS Journal 273 (2006) 2357–2373 ª 2006 The < /b> Authors Journal compilation ª 2006 FEBS P R Vajjhala et al Role of the < /b> Vps4 b domain Table 2 Primers used for mutagenesis Primer Sequence Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 Vps4 5¢-CGCTGCAGTAAGAGCAGTAAACCCG-3¢ 5¢-GAGAATCAGTGTCGACTTCATCTATAAAAATAATAGAAGGTTTATT-3¢ . 5¢-AGCACGTTAATCAATTGACTAATCTGGTTCTTTTAA-3¢ Vps4 RDE F 5¢-AAGCAAGAACAGTTCACTGCAGCTTTTGGTCAAGCAGGTAACTAGTCAATTGAT-3¢ Vps4 RDE R 5¢-ATCAATTGACTAGTTACCTGCTTGACCAAAAGCTGCAGTGAACTGTTCTTGCTT-3¢ Vps4. 5¢-GAGAATCAGTGTCGACTTCATCTATAAAAATAATAGAAGGTTTATT-3¢ Vps4 SalIF 5¢-GCCCATATTCGTCGACGCGCTAACAGGTACCAGAGGAGAAGGAGAGAGCGAAGCAAGTAG-3¢ Vps4 Dstr R 5¢-GGGCGGATCCTCTGCTTTTCTTTATC-3¢ Vps4