Báo cáo khoa học: Activation of the plasma membrane H+-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H+-ATPase–acetylated tubulin complex docx

Activation of the plasma membrane H + -ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H + -ATPase–acetylated tubulin complex Alexis N. Campetelli 1 , Gabriela Previtali 1 , Carlos A. Arce 2 ,He ´ ctor S. Barra 2 and Ce ´ sar H. Casale 1 1 Departamento de Biologı ´ a Molecular, Facultad de Ciencias Exactas, Fı ´ sico-Quı ´ micas y Naturales, Universidad Nacional de Rı ´ o Cuarto, Co ´ rdoba, Argentina 2 Centro de Investigaciones en Quı ´ mica Biolo ´ gica de Co ´ rdoba (CIQUIBIC), UNC-CONICET, Departamento de Quı ´ mica Biolo ´ gica, Facultad de Ciencias Quı ´ micas, Universidad Nacional de Co ´ rdoba, Argentina We recently described the interaction of Na + ⁄ K + -ATP ase with acetylated tubulin in neural [1–3] and non- neural cells [4]. Formation of such a complex inhibits ATPase activity. Conversely, dissociation of the com- plex leads to activation of the enzyme. The ATPase– acetylated tubulin complex behaves as a hydrophobic Keywords glucose; H + -ATPase; proton pump; tubulin; yeast Correspondence H. S. Barra, Departamento de Quı ´ mica Biolo ´ gica, Facultad de Ciencias Quı ´ micas, Universidad Nacional de Co ´ rdoba, Ciudad Universitaria, 5000-Co ´ rdoba, Argentina Fax: +54 3514334074 Tel: +54 3514334168 E-mail: hbarra@dqb.fcq.unc.edu.ar (Received 29 July 2005, revised 2 September 2005, accepted 6 September 2005) doi:10.1111/j.1742-4658.2005.04959.x In the yeast Saccharomyces cerevisiae, plasma membrane H + -ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usu- ally displays hydrophilic properties, but behaves as a hydrophobic com- pound when complexed with H + -ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H + -ATP- ase–tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H + -ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H + -pumping activity. The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, sug- gesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentra- ted near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H + -ATPase) immu- noglobulins indicated a physical interaction between H + -ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Dou- ble immunofluorescence, observed by confocal microscopy, indicated that H + -ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H + -ATPase–tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H + -ATPase activity by acetylated tubulin and concomitant increase of the H + -ATP ase–tubulin complex. Abbreviations HAT, hydrophobic acetylated tubulin. 5742 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS compound, whereas free tubulin is soluble in water. This property allowed us to isolate the complex, termed hydrophobic acetylated tubulin (HAT), which was subsequently quantified by immunoblot with antibody specific to acetylated tubulin. The present study is focused on the H + -ATPase of yeast plasma membrane, another P-type ATPase. This enzyme is formed by several polypeptides, most prom- inently a 100 kDa chain that is partly immersed in the plasma membrane. It functions to hydrolyze ATP and to transport H + out of the cell, thereby regulating internal pH. An important finding was that glucose activates the plasma membrane H + -ATPase of yeast cells [5]. This activation has been extensively investi- gated; however, its molecular mechanism is not com- pletely understood. The activation of yeast H + -ATPase by glucose is regulated at the transcriptional and post-transcrip- tional levels [6–10]. Glucose increases mRNA synthesis by inducing transcription of the H + -ATPase gene (PMA1p), increases phosphorylation of the enzyme, the K m decreases and the V max increases. Proteolytic degradation of a protein that inhibits the glucose- activation process also seems to be involved [11]. We report here that yeast H + -ATPase interacts with tubu- lin, that such an interaction inhibits enzyme activity, and that glucose treatment of cells induces dissociation of the ATPase–tubulin complex with a concomitant increase in the amount of active enzyme. Results Effect of D-glucose on H + -ATPase activity and HAT quantity Yeast cells suspended in Mes-Tris buffer were incuba- ted for 20 min at 30 °C in the presence of various concentrations of d-glucose. Plasma membranes were isolated, and H + -ATPase activity and HAT quantity were measured. d-glucose produced concentration- dependent activation of H + -ATPase, as measured by its ATP-hydrolyzing capacity, reaching a plateau at 150 lm (Fig. 1A). At this concentration, HAT was decreased by 80%. Activation of plasma H + -ATPase by d-glucose is a very rapid process; when the yeast cells were incubated in unbuffered medium in the pres- ence of 1 mmd-glucose, the level of HAT decreased to almost zero in less than 5 min (Fig. 1B). Acidification of the medium was observed, with a minimal pH value reached in less than 10 min. The ATP-hydrolysis capa- city of H + -ATPase also increased quickly. It should be noted that dissociation of the tubulin–H + -ATPase complex (initial rate of decay of HAT) precedes enzyme activation determined by its H + -pumping or ATP hydrolyzing activity (Fig. 1B). These rapid effects of d-glucose suggest action at the membrane level, poss- ibly during the transport of glucose into the cell. We therefore studied the effect of 2-deoxy-d-glucose, a competitive substrate for glucose uptake [12]. This AB Fig. 1. Effect of D -glucose on H + -ATPase activity and hydrophobic acetylated tubulin (HAT) quantity in the plasma membrane of Saccharomy- ces cerevisiae. (A) Glucose-starved yeast cells were incubated for 20 min at 30 °C in Mes ⁄ Tris buffer in the presence of D-glucose at the indicated concentrations. (B) Yeast cells were incubated at 30 °C in physiological solution in the presence of 1 m MD-glucose for the indica- ted times. Cells were processed for membrane isolation and subsequent determination of H + -ATPase activity and HAT quantity, as des- cribed in the Experimental procedures. (B) In a parallel experiment, at the indicated time-points the external pH was measured instead of proceeding to membrane isolation. Data represent the mean ± SD from three independent experiments. Acetylated tubulin bands shown in the upper panels are from a representative experiment. A. N. Campetelli et al. Dissociation of tubulin–H + -ATPase complex by glucose FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5743 compound, at a concentration of 1 mm in the culture medium, blocked the H + -ATPase activation and HAT decrease induced by 30 min treatment of cells with 0.1 mmd-glucose (Table 1, Exp. 1); however, it had no effect when used at 10 mm. The blocking of the ATP- ase activating effect of glucose was not the result of a toxic effect of 2-deoxy-d-glucose, as subsequent incuba- tion in the presence of 10 mm glucose resulted again in H + -ATPase activation with concomitant dissociation of the ATPase–tubulin complex (HAT decrease) (Table 1, Exp. 2). The blocking effect of 2-deoxy-d- glucose is immediate as, 1 min after its addition, the H + -pumping activity of H + -ATPase ceased (Fig. 2A) and the HAT quantity began to increase (Fig. 2B). When isolated plasma membranes from glucose- starved yeast were used instead of intact cells, treat- ment with 1 mmd-glucose for 20 min had no effect on the ATP-hydrolyzing capacity of H + -ATPase, nor on HAT quantity (data not shown). Previous studies [2–4], and the findings described below, suggest that the decrease of HAT in the plasma membrane should be interpreted as a lower level of the H + -ATPase–tubulin complex. We therefore presume that the immediate uptake of glucose after its addition induces dissociation of the H + -ATPase–acetylated tub- ulin complex, resulting in an increased enzyme activity, characterized by higher ATPase hydrolyzing capacity and H + -pumping activity. Characterization of acetylated tubulin in Saccharomyces cerevisiae Localization of acetylated tubulin in cells was studied by immunofluorescence. Acetylated tubulin (Fig. 3A) Table 1. Effect of 2-deoxy-D-glucose on H + -ATPase activation induced by D-glucose. Glucose-starved cells suspended in Mes-Tris buffer were incubated at 37 °C under the indicated conditions for 30 min (experiment 1) or 60 min (experiment 2), then frozen in liquid nitrogen, and hydrophobic acetylated tubulin (HAT) and H + -ATPase activities were measured. Values represent the mean ± SD from three independ- ent experiments. Experiment Additions and 30 min of incubation Additions and 30 min of further incubation HAT (% of control) H + -ATPase activity (lmol PiÆmin )1 Æmg protein )1 ) 1 No addition (control) 100 ± 11 1.49 ± 0.6 0.1 m MD-glucose 20 ± 7 8.42 ± 0.27 0.1 m MD-glucose plus 82 ± 9 2.21 ± 0.25 1m M 2-deoxy-D-glucose 10 m MD-glucose plus 29 ± 13 5.72 ± 0.5 1m M 2-deoxy-D-glucose 2 No addition (control) No addition 100 ± 9 1.39 ± 0.5 0.1 m MD-glucose plus 1m M 2-deoxy-D-glucose 10 m MD-glucose 28 ± 12 5.60 ± 0.7 A B Fig. 2. Effect of 2-deoxy-D-glucose on H + -pumping activity and hydrophobic acetylated tubulin (HAT) quantity of Saccharomyces cerevisiae. (A) Yeast cells were incubated at 30 °C in physiological solution in the presence of 1 m MD-glucose, and the external pH was measured at the indicated time-points. At the time indicated by the arrow, 2-deoxy- D-glucose (10 mM final concentration) was added (d). A control, with- out added 2-deoxy- D-glucose, was included (s). (B) In a parallel experiment, instead of pH measurement, HAT quantity was measured. Data represent the mean ± SD from three independent experiments. Acetylated tubulin bands shown are from a representative experiment. Dissociation of tubulin–H + -ATPase complex by glucose A. N. Campetelli et al. 5744 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS and H + -ATPase (Fig. 3C) are localized near the plasma membrane, whereas a-tubulin (Fig. 3B) is dis- tributed uniformly throughout the cell. We showed previously [13,14] that the presence of acetylated tubulin in membrane preparations from mammalian cells is the result not of an intrinsic prop- erty of the protein, but because of its association with an integral membrane protein subsequently identified as Na + ⁄ K + -ATPase [1]. Acetylated tubulin present in the membrane was released by incubation at alkaline pH (0.1 m Na 2 CO 3 , pH 11.5) and the remaining mem- branes (depleted of acetylated tubulin) could again accept exogenously added acetylated tubulin [14]. We therefore investigated the biochemical properties of acetylated tubulin in yeast plasma membrane, and found them to be similar to those of acetylated tubulin in mammalian cells. After alkaline treatment, yeast plasma membrane was depleted of total tubulin (deter- mined with DM1A antibody) as well as acetylated tubulin (determined with 6-11B-1 antibody) (Fig. 4, lane 2 vs. lane 1). When acetylated tubulin-depleted membranes were incubated in the presence of acetylat- ed tubulin (isolated from rat brain), this protein was bound to the membrane fraction (lane 3) and subse- quently removed by alkaline treatment (lane 4). These results indicate that acetylated tubulin in yeast Fig. 3. Immunofluorescent localization of total microtubules, acetyl- ated tubulin, and H + -ATPase in Saccharomyces cerevisiae. Sphero- plasts from glucose-starved yeast cells were fixed on coverslips and stained with antibody against acetylated tubulin (A), total a-tub- ulin (B), or H + -ATPase (C), using corresponding secondary antibod- ies conjugated to fluorescein for acetylated and total a-tubulin and rhodamine for H + -ATPase. Fig. 4. Removal of acetylated tubulin from yeast membranes by alkaline treatment. Plasma membranes (5 mg of protein) from yeast (lane 1) were suspended in 5 mL of 0.1 M Na 2 CO 3 , pH 11.5, and incubated for 20 min at 4 °C. The sample was centrifuged at 100 000 g for 20 min, and the pellet (lane 2) was resuspended in Mes ⁄ Tris buffer, pH 6.5. This preparation (2 mg of protein) was incubated for 20 min at 37 °C with partially purified brain tubulin (2 mg) containing a high proportion of acetylated isotype. The pre- paration was centrifuged at 100 000 g for 20 min, and the pellet (lane 3) was collected. The pellet was treated with alkaline solution as described above, and centrifuged to sediment the membrane fraction (lane 4). The starting plasma membrane from yeast, and the subsequent pellets, were processed to determine hydrophobic acetylated tubulin (HAT) by immunoblot, as described in the Experi- mental procedures, using antibody to total a-tubulin (left panel) and to acetylated tubulin (right panel). A. N. Campetelli et al. Dissociation of tubulin–H + -ATPase complex by glucose FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5745 membrane is a peripheral protein that is bound to some integral membrane protein, presumably H + -ATP- ase, in a manner similar to its association with Na + ⁄ K + -ATPase in the plasma membrane of mamma- lian cells. To confirm the interaction of acetylated tubulin with H + -ATPase and the presence of the H + -ATPase–acet- ylated tubulin complex in membranes of glucose-starved yeast, we performed immunoprecipitation experiments using either anti-(acetylated tubulin) immunoglobulin or anti-(H + -ATPase) immunoglobulin bound to Seph- arose beads. When detergent-solubilized plasma mem- branes from glucose-starved yeast were treated with anti-(acetylated tubulin)–Sepharose beads and then cen- trifuged, in addition to tubulin, the pellet contained the 110 kDa H + -ATPase polypeptide (Fig. 5B, lane –). When solubilized membranes were treated with anti- H + -ATPase–Sepharose beads, the pellet contained acet- ylated tubulin in addition to the 110 kDa H + -ATPase polypeptide (Fig. 5C, lane –). These findings indicate that a complex containing both acetylated tubulin and H + -ATPase is present in solubilized yeast membranes from glucose-starved cells. When cells were pretreated with 1 mm glucose for 1 h and then processed for im- munoprecipitation with anti-H + -ATPase–Sepharose beads, the pellet contained the 110 kDa H + -ATPase polypeptide and only a minor amount of acetylated tub- ulin (Fig. 5C, lane +), indicating that glucose treatment induced dissociation of the H + -ATPase–acetylated tub- ulin complex. An identical conclusion was drawn from immunoprecipitation experiments with anti-(acetylated tubulin)–Sepharose beads (Fig. 5B, lane +). Figure 5D shows that Sepharose beads without bound antibodies practically do not adsorb tubulin or ATPase. To esti- mate the proportion of acetylated tubulin and H + -ATP- ase that was immunoprecipitated, we determined also the amount of each protein present in the solubilized- membrane preparations before immunoprecipitation (Fig. 5A). From visual comparison of blots, it can be seen that most of the acetylated tubulin and ATPase were involved in the immunoprecipitation process. By measuring the intensity of the ATPase band in Fig. 5B (lane –) with respect to that in Fig. 5A (lane –), it was calculated that 88 ± 15% of ATPase was associated with acetylated tubulin. In addition, by comparing the intensity of the ATPase band in lane (+) with respect to that in lane (–) in Fig. 5B, it was calculated that 88 ± 12% of the complex was dissociated by glucose treatment. These results represent the mean ± SD val- ues from three independent experiments. Further evidence for the occurrence of the H + -ATP- ase–acetylated tubulin complex in the plasma mem- brane of S. cerevisiae was the co-localization of the two components in confocal immunofluorescence ana- lysis. The image obtained with anti-(acetylated tubulin) immunoglobulin partially overlapped that obtained with anti-(H + -ATPase) immunoglobulin (Fig. 6). Enlargement of a portion of the merge image (Fig. 6D) shows that H + -ATPase (red) is localized on the plasma membrane without extending into the cyto- plasm, while acetylated tubulin (green) is localized near the membrane, overlapping (yellow) with the inner side of the region occupied by H + -ATPase. The more external region of the membrane H + -ATPase is not in contact with acetylated tubulin. When cells were pre- treated with 1 mm glucose for 1 h and then processed for immunofluorescence, acetylated tubulin was distri- buted uniformly throughout the cytoplasm and did not Fig. 5. Physical interaction between acetylated tubulin and H + -ATP- ase in Saccharomyces cerevisiae membrane. (A) To estimate the total amount of both acetylated tubulin and ATPase present in the detergent-solubilized yeast plasma membrane from cells previously treated (lanes +) or not treated (lanes –) with 1 m M glucose, 23 lL aliquots were subjected to SDS ⁄ PAGE and simultaneous immuno- blot staining with anti-(acetylated tubulin) and anti-(H + -ATPase) immunoglobulin. (B) and (C) An aliquot of 0.7 mL of detergent-solubi- lized yeast plasma membrane from cells previously treated (lanes +) or not treated (lanes –) with 1 m M glucose was mixed with 0.3 mL of packed anti-acetylated tubulin (6-11-B-1)–Sepharose beads (B), or anti-H + -ATPase (Pma1p)–Sepharose beads (C), and incubated at 20 °C for 30 min. The samples were then centrifuged, and the preci- pitated material was washed five times with 50 m M Tris ⁄ HCl buffer, pH 7.4, containing 150 m M NaCl (TBS)-Triton. A fraction of 50 lLof packed beads was resuspended in 50 lL of Laemmli sample buffer [13], treated at 50 °C for 15 min, and centrifuged. Twenty microlitre aliquots of the soluble fractions were subjected to SDS ⁄ PAGE and then to simultaneous immunoblot staining with anti-(acetylated tubu- lin) and anti-(H + -ATPase) immunoglobulin. (D) A control was run in parallel using glycine–Sepharose instead of antibodies–Sepharose. Note that the protein bands shown arise from equal amounts of membrane preparations. Dissociation of tubulin–H + -ATPase complex by glucose A. N. Campetelli et al. 5746 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS overlap ATPase, which remained concentrated at the periphery. This is further evidence that glucose induced dissociation of the H + -ATPase–acetylated tubulin complex. H + -ATPase activity of isolated plasma membrane is inhibited by tubulin Plasma membranes isolated from S. cerevisiae were incubated in the presence of various amounts of tubu- lin purified from rat brain, and H + -ATPase activity was determined. Enzyme activity decreased as the tub- ulin concentration increased (Fig. 7A). Two prepara- tions were used containing proportions of acetylated tubulin isotype that differed approximately fourfold. The amount of tubulin required to obtain 50% inhibi- tion of H + -ATPase was approximately fourfold higher for the low-proportion preparation. For each tubulin concentration, we determined the amount of acetylated tubulin that was converted into hydrophobic com- pound (HAT) (Fig. 7B). Such conversion reflects for- mation of the tubulin–ATPase complex. The HAT quantity increased as the tubulin concentration increased. Taken together, these results indicate that the association of H + -ATPase with acetylated tubulin inhibits enzyme activity. Discussion In yeast, the H + -ATPase activity of the plasma mem- brane is up-regulated by external glucose [5–10]. We showed, in this study, that tubulin interacts with H + -ATPase to form a complex in which the enzyme is inhibited, and that treatment of the cells with glucose dissociates the complex and restores enzyme activity. Although tubulin is a hydrophilic protein, it behaves as a hydrophobic compound when it interacts with H + -ATPase. The complex can therefore be isolated by detergent-partitioning with Triton X-114. The hydro- phobic tubulin partitioning into the detergent phase (HAT) is the acetylated tubulin forming a complex with H + -ATPase. The finding of the H + -ATPase–acetylated tubulin complex in plasma membranes of glucose-starved yeast cannot be attributed to an in vitro artifact (i.e. associ- ation of acetylated tubulin with H + -ATPase during the isolation of HAT) because a high vs. a low content of HAT is measured in glucose-untreated or -treated cells, respectively, by the same isolation procedure. If the interaction between tubulin and H + -ATPase were established during the in vitro procedure, the amount of HAT in membranes from glucose-treated cells would be the same as that from glucose-untreated cells. The possibility that the presence of glucose dur- ing the in vitro procedure for measuring HAT induced dissociation of the ATPase–acetylated tubulin complex was also experimentally ruled out. The treatment of isolated membranes with 1 mm glucose did not Fig. 6. Colocalization of acetylated tubulin and H + -ATPase in Sac- charomyces cerevisiae. Yeast cells were treated (+ glucose) or were not treated (– glucose) with 1 m M glucose for 1 h and proc- essed to obtain spheroplasts, which were then fixed on coverslips and subjected to double immunofluorescence using antibodies spe- cific to H + -ATPase (A and A¢) and to acetylated tubulin (B and B¢). (C) and (C¢) Merge image. (D) and (D¢) Enlargement of the area indi- cated by a rectangle in (C) and (C¢), respectively. A. N. Campetelli et al. Dissociation of tubulin–H + -ATPase complex by glucose FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5747 decrease HAT or increase H + -ATPase activity (data not shown). The presence of the H + -ATPase–acetylated tubulin complex in the plasma membrane of glucose-starved yeast is also supported by results from immunoprecipi- tation experiments (Fig. 5). As acetylated tubulin pre- sent in detergent-solubilized membranes isolated from glucose-starved yeast was precipitated by treatment with anti-(H + -ATPase) immunoglobulin (Fig. 5C, lane –), and H + -ATPase was precipitated by treatment with anti-(acetylated tubulin) immunoglobulin (Fig. 5B, lane –), we conclude that a complex containing H + - ATPase and acetylated tubulin is present in the plasma membrane. Colocalization of acetylated tubulin with H + -ATPase in the periphery of the cell was observed by confocal microscopy (Fig. 6). Although the overlap was partial, it is clear that some part of the acetylated tubu- lin shares space with some part of the H + -ATPase, rein- forcing the idea of a complex between H + -ATPase and acetylated tubulin at the plasma membrane. This com- plex was dissociated by pretreatment of cells with glu- cose. In fact, when cells were treated with 1 mm glucose for 1 h prior to immunoprecipitation and immunofluo- rescence experiments, acetylated tubulin was diminished in isolated membranes (Fig. 5A, lane +), and practi- cally was not precipitated by anti-(H + -ATPase) immu- noglobulin (Fig. 5C, lane +), and ATPase and acetylated tubulin did not colocalize (Fig. 6D¢). We observed that treatment of cells with glucose induces dissociation of the complex [seen as a decrease of HAT, a decrease of acetylated tubulin immuno- precipitated by anti-(H + -ATPase) immunoglobulin, a decrease of H + -ATPase immunoprecipitated by anti- tubulin immunoglobulin, and a loss of colocalization] with concomitant increase of H + -ATPase activity. This provides strong evidence for existence of the H + -ATPase–acetylated tubulin complex in the plasma membrane of the yeast cell, and for a regulatory role of tubulin on activity of the enzyme. We often refer to ‘H + -ATPase–tubulin’ rather than ‘H + -ATPase–acetylated tubulin’ complex, for conveni- ence. In fact, it is quite possible that acetylated tubulin is the only tubulin isotype that forms the complex, as H + -ATPase activity is more strongly inhibited when the tubulin preparation contains a higher proportion of the acetylated isotype (Fig. 7). We do not know whether molecules other than H + -ATPase and tubulin are also part of the complex. Although the inhibition of H + -ATPase activity observed when the complex is formed suggests direct interaction of tubulin with ATPase, we cannot rule out the possibility that other molecules mediate this interac- tion. Involvement of glucose transporters is a reason- able assumption because the complex is dissociated when glucose is transported into the cell, but not when glucose is added to previously isolated membranes (data not shown). This possibility is supported by the obser- vation that 2-deoxy-d-glucose, a competitive substrate for glucose uptake, suppressed activation of glucose and dissociation of the ATPase–tubulin complex in a con- centration-dependent manner (Table 1). It is possible that the dissociation of the acetylated tubulin–H + - A B Fig. 7. Effect of exogenous tubulin on H + -ATPase activity and hydrophobic acetylated tubulin (HAT) quantity in isolated membranes. Plasma membrane (70 lg of proteinÆmL )1 ) isolated from glucose-treated Saccharomyces cerevisiae was incubated at 37 °C for 25 min in a final vol- ume of 1 mL of Mes-Tris ⁄ phenylmethanesulfonyl fluoride buffer, pH 6.5, in the presence of various amounts of rat brain tubulin preparations containing low (d)orhigh(s) levels of the acetylated isotype (for details see the Experimental procedures). After incubation, appropriate aliquots were removed to determine H + -ATPase activity (A) and HAT quantity (B). Values represent the mean ± SD from three independent experiments. H + -ATPase activity in the absence of added tubulin was 8.2 ± 0.3 lmol PiÆmin )1 Æmg )1 protein. Acetylated tubulin bands shown are from a representative experiment. Dissociation of tubulin–H + -ATPase complex by glucose A. N. Campetelli et al. 5748 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS ATPase complex requires the presence of Snf3p (a glu- cose sensor), Gpa2 protein (a G protein) [10] and protein kinases [15], which were demonstrated to parti- cipate in the glucose-induced activation of the plasma membrane ATPase. Our results in the present work show that a glucose- sensitive association ⁄ dissociation of acetylated tubulin and H + -ATPase participates in an early stage of the mechanism that leads to, respectively, inhibition and activation of the enzyme. The increase of enzyme activity (determined by ATP-hydrolyzing capacity or by H + -pumping activity) starts immediately after glu- cose addition and reaches a maximum in  10 min, whereas dissociation of the ATPase–tubulin complex (decrease of HAT quantity) is completed within the first 2 min (Fig. 1B, and Fig. 2A,B). The reason for this temporal difference is not clear. It is possible that besides dissociation of the complex, the enzyme requires some additional, time-consuming process for its activation. Anyway, the important conclusion from these experiments is that dissociation of the ATPase– tubulin complex is at least part of the reason for the increased enzyme activity induced by external glucose. In agreement with this view, exogenously added tubu- lin was bound to membrane H + -ATPase and inhibited enzyme activity (Fig. 7), indicating that H + -ATPase activation by glucose is caused by an increased concen- tration of active enzyme. Similar effects of tubulin were demonstrated for Na + ⁄ K + -ATPase in our previ- ous studies [2–4]. Interestingly, for both H + -ATPase in yeast and Na + ⁄ K + -ATPase in mammalian cells [3,4], the ATPase–acetylated tubulin complex can be dissociated during the uptake of d-glucose and l-glutamate, respectively. In either case, corresponding enzyme activity increases upon dissociation of the complex. We suspect that these events are part of a signal trans- duction cascade and are accordingly investigating the nature of membranous and cytoplasmic components of the system, interactions between components and modulation of these interactions. Experimental procedures Materials Triton X-114, ATP, d-glucose, 2-deoxy-d-glucose, mouse monoclonal antibody DM1A specific to a-tubulin, mouse monoclonal antibody 6-11B-1 specific to acetylated tubulin, anti-mouse and anti-rabbit IgG conjugated to peroxidase were from Sigma Chemical Co. (St Louis, MO, USA). [ 32 P]ATP[cP] was from Perkin-Elmer (Wellesley, MA, USA). Rabbit polyclonal antibody Pma1p, specific to H + -ATPase, was provided by R. Serrano (Instituto de Bio- logı ´ a Molecular y Celular de Plantas, Valencia, Spain). Yeast strain and growth conditions S. cerevisiae, strain CECT 1891 (Spanish Type Culture Collection, University of Valencia, Valencia, Spain), was used. Cells were grown on synthetic medium YP [0.5% (w ⁄ v) yeast extract and 0.5% (w ⁄ v) peptone] containing 4% (w ⁄ v) glucose. Cells were grown in a rotary incubator (New Brunswick model G24; 200 r.p.m.; NJ, USA) at 30 °C until the end of the exponential phase. Cells were then harvested (centrifugation at 1000 g for 10 min), sus- pended in Mes ⁄ Tris buffer (100 mm Mes ⁄ Tris, pH 6.5) and magnetically stirred for 60 min to eliminate glucose activation [5]. These cells are referred to as ‘glucose- starved cells’. Yeast plasma membrane preparation Plasma membranes were isolated by a modification of the method of Villalba et al. [16]. Briefly, yeast cells were suspen- ded in Mes ⁄ Tris buffer supplemented with 1 mm phenyl- methanesulfonyl fluoride (Mes ⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer), with or without d-glucose, according to the conditions of each experiment, homogenized by vigorous shaking with glass beads, and centrifuged at 1000 g for 10 min. The resulting supernatant was centrifuged at 70 000 g for 60 min to obtain the total membrane fraction. The total membrane fraction from 10 g of cells was suspen- ded in 3 mL of Mes ⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer. The suspension was applied to a discontinuous gradi- ent – from 5.0 mL of 60% (w ⁄ v) sucrose to 5.0 mL of 40% (w ⁄ v) sucrose – in 10 mm Tris ⁄ HCl buffer (pH 7.6) contain- ing 1 mm EDTA and 1 mm dithiothreitol. Plasma mem- branes were centrifuged for 3 h at 100 000 g, collected from the 40 ⁄ 60% sucrose interface, diluted 10-fold with Mes ⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer and centri- fuged at 100 000 g for 1 h. The resulting pellet was resus- pended in Mes ⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer and stored at )70 °C until use (maximum storage time 3 months). Plasma membrane H + -ATPase activity assay We used the [ 32 P]ATP[cP] hydrolysis method of Malpartida & Serrano [6]. The incubation mixture (0.5 mL final vol- ume) contained Mes ⁄ Tris ⁄ phenylmethanesulfonyl fluoride buffer, 10 mm MgCl 2 ,2mm [ 32 P]ATP[cP] (450 d.p.m.Æ nmol )1 ) and 7 lgÆmL )1 plasma membrane protein. After 20 min at 30 °C, the reaction was stopped by adding 50 lL of 66% (w ⁄ v) trichloroacetic acid per mL of incubation mixture, and the released 32 P i was quantified. Plasma mem- brane H + -ATPase activity was calculated as the difference A. N. Campetelli et al. Dissociation of tubulin–H + -ATPase complex by glucose FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS 5749 of ATP hydrolysis in the presence vs. absence of 100 l m sodium orthovanadate. Isolation and determination of HAT HAT was isolated into the Triton X-114 phase, as des- cribed previously [2], except that plasma membranes were solubilized with Triton X-100, as described below, before adding Triton X-114 and partitioning. Membranes from S. cerevisiae (28 lg of protein) were washed once with 50 mm Tris ⁄ HCl buffer, pH 7.4, containing 150 mm NaCl (TBS) and immediately solubilized in 1 mL of TBS con- taining 1% (v ⁄ v) Triton X-100. After 30 min at 0 °C, the preparation was centrifuged at 100 000 g for 15 min, and the supernatant fraction was processed for HAT isolation by partitioning in Triton X-114 (1% final detergent con- centration). For phase separation, the preparation was warmed at 37 °C for 5 min and centrifuged at 600 g for 5 min. The aqueous upper phase and detergent-rich lower phase were carefully separated, and the detergent- rich phase (which contains HAT) was washed once with TBS. Aliquots were subjected to electrophoresis and immunoblotting to determine acetylated and total tubulin. Electrophoresis and immunoblotting Proteins were separated by SDS ⁄ PAGE on 10% (w ⁄ v) polyacrylamide slab gels [17], transferred to nitrocellulose, and reacted with mouse monoclonal antibody 6-11B-1 (dilution 1 : 1000) to determine acetylated tubulin [18], mouse monoclonal antibody DM1A (dilution 1 : 1000) to determine total a-tubulin, or rabbit polyclonal antibody Pma1p (dilution 1 : 1000) to determine plasma membrane H + -ATPase [15]. The nitrocellulose sheet was reacted with anti-mouse (for 6-11B-1 and DM1A antibodies) or anti- rabbit (for Pma1p antibody) IgG conjugated with peroxi- dase. Intensities of tubulin bands were quantified by Scion imaging software. Measurement of H + -pumping activity Samples of cells ( 50 mg) were washed twice, suspended in 15 mL of 0.9% (w ⁄ v) NaCl, and stirred gently at 30 °C in a beaker. Incubation conditions were as indicated in each experiment. External pH was measured using a pH meter with a calomel electrode. Preparation of spheroplasts from yeast S. cerevisiae cells (50 mg fresh weight) were washed twice in 2 mL of 0.1 m Tris ⁄ HCl, pH 7.2, containing 5 mm EGTA and 5 mm dithiothreitol, incubated with stirring for 10 min, washed with water (containing 1 mm dithio- threitol), and resuspended in 2 mL of 0.1 m Tris ⁄ HCl, pH 7.2, containing 1 m sorbitol and 1 mm dithiothreitol. Zymolyase was added to the suspension (final concentra- tion 0.1 mgÆmL )1 ), gently stirred for 30 min, and cells were harvested by centrifugation. The pellet was resus- pended in 2 mL of 0.1 m Tris ⁄ HCl, pH 7.2, containing 5mm EGTA and 5 mm dithiothreitol, and used immedi- ately. All procedures were carried out at room tempera- ture. Immunofluorescence Spheroplasts were fixed with anhydrous methanol at )20 °C on coverslips. Samples were washed, incubated with 2% (w ⁄ v) BSA in NaCl ⁄ P i (PBS) for 30 min, and stained by indirect immunofluorescence, as described by DeWitt et al. [19]. Two primary antibodies were used: mouse 6-11B-1 monoclonal antibody (diluted 1 : 200) to visualize acetylated tubulin, and rabbit Pma1p polyclonal antibody (diluted 1 : 200) to determine plasma membrane H + -ATPase. Fluorescein-conjugated anti-mouse IgG and Rhodamine-conjugated anti-rabbit immunoglobulin, at a 1 : 400 dilution, were used as secondary antibodies, respectively. Coverslips were mounted in FluorSave and observed with an LSM 5 Pascal confocal microscope (Zeiss, Jena, Germany) using dual channel filters for sim- ultaneous viewing of rhodamine and fluorescein isothio- cyanate fluorochromes. Tubulin preparations containing different proportions of acetylated tubulin The procedure used to isolate rat brain tubulin prepara- tions containing different proportions of the acetylated iso- type has been described previously [2]. These preparations contained low and high acetylated tubulin proportions dif- fering by a factor of  4. Preparation of 6-11B-1–Sepharose and Pma1p– Sepharose 6-11B-1 and Pma1p antibodies were covalently bound to cyanogen bromide-activated Sepharose 4B following the procedure of Hubert et al. [20] with slight modifications. Sepharose beads were washed with a 100-fold volume excess of 0.001 m HCl at 21 °C. The resulting packed beads (0.3 mL) were mixed with 2 mg of 6-11B-1 antibody (or 2 mg of Pma1p antibody) in 1 mL of coupling buffer (0.5 m NaCl containing 0.2 m NaHCO 3 , pH 8.2). The mix- ture was agitated on a platform rocker for 4 h at 21 °C, and loaded into a small chromatographic column. Unbound 6-11B-1 antibody (or Pma1p antibody) was removed by filtration and by washing with 10 mL of coup- ling buffer. The 6-11B-1-Sepharose (or Pma1p-Sepharose) Dissociation of tubulin–H + -ATPase complex by glucose A. N. Campetelli et al. 5750 FEBS Journal 272 (2005) 5742–5752 ª 2005 FEBS beads were loaded into a beaker, and added with 2 mL of coupling buffer containing 0.2 m glycine to block unreacted Sepharose sites. The mixture was agitated for 2 h at 21 °C, and unbound glycine was removed by washing the beads with 20 mL of coupling buffer. The resulting affinity adsorbant was washed with 30 mL of 0.01 m Tris ⁄ HCl, pH 8, containing 0.14 m NaCl and 0.025% NaN 3 , and stored at 4 °C until use (maximum 2 days). Protein determination Protein concentration was determined by the Bradford method [21]. Acknowledgements We thank Dr J. A. Curtino for critical reading of the manuscript, Dr R. Serrano (Valencia, Spain) for the generous gift of anti-(H + -ATPase), and Dr S. 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