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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02749
Research Article |
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109, USA
* Author for correspondence (e-mail: amychang{at}umich.edu)
Accepted 19 October 2005
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Key words: Ubiquitylation, Quality control, Plasma membrane ATPase
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). It has been proposed that monoubiquitylated substrates are recognized by components of the internalization machinery, such as epsin and Eps15 via their ubiquitin-binding modules. It has also been proposed that ubiquitylation participates in a quality control mechanism to remove misfolded proteins from the surface (Sharma et al., 2004). In contrast to proteins that undergo rapid internalizaton, the prolonged stability of some cell-surface components is crucial because they have a structural role or because their function is required under all environmental conditions.
In the yeast Saccharomyces cerevisiae, the plasma membrane ATPase, Pma1, serves as a paradigm for stability at the plasma membrane. Pma1 is an abundant polytopic membrane protein with ten transmembrane domains and it has remarkable longevity with a half-life of 11 hours (Benito et al., 1991). Normally, Pma1 is not ubiquitylated (Wang and Chang, 2002). The persistence of Pma1 at the cell surface reflects its major role in generating the membrane potential that is essential for growth (Morsomme et al., 2000). How Pma1 acquires stability at the cell surface is not clear. Pma1 is associated with lipid rafts – microdomains enriched in sphingolipids and cholesterol (Bagnat et al., 2000) – and it has been suggested that its recruitment to detergent-insoluble glycolipid-enriched complexes (DIGs) participates in promoting Pma1 stability. Supporting this idea is the observation that Pma1 turnover is increased when sphingolipid synthesis is impaired in lcb1-100 cells (Wang and Chang, 2002).
In a previous study, we described a temperature-sensitive mutant, pma1-10, impaired in Pma1 stability at the cell surface (Gong and Chang, 2001). Pma1-10 has two changes, A165G and V197I, predicted to reside in the first cytoplasmic loop between transmembrane segments 2 and 3 of Pma1. At the restrictive temperature, newly synthesized Pma1-10 undergoes rapid internalization from the cell surface and delivery for degradation in the vacuole. When Pma1-10 is the sole copy of Pma1, cells cannot grow at 37°C. In contrast to wild-type Pma1, Pma1-10 has increased sensitivity to trypsin (suggesting it is misfolded), is hypophosphorylated, and fails to associate with DIGs (suggesting exclusion from lipid rafts) (Gong and Chang, 2001). In this study, we have investigated two possible mechanisms for rapid removal of mutant Pma1 from the cell surface. One possibility is that Pma1-10 misfolding leads to a failure in the production of stabilizing interactions at the plasma membrane, including phosphorylation and DIG inclusion. Alternatively, Pma1-10 is recognized by a quality control mechanism and targeted for removal. Our results suggest that internalization of Pma1-10 is promoted by its ubiquitylation occurring at or before its arrival at the plasma membrane. Internalization is dependent on components of the endocytic machinery, perhaps via their recognition and association with ubiquitin. However, ubiquitylation is reversible: when Pma1-10 is sequestered at the plasma membrane by a block in endocytosis, increased phosphorylation and DIG association accompany deubiquitylation of Pma1-10. We suggest rapid internalization of mutant Pma1 may preempt stabilizing interactions.
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). By shifting cells to methionine-free medium, synthesis of HA-Pma1-10 is derepressed, but growth is unaffected because the cells have a chromosomal copy of wild-type Pma1. Indirect immunofluorescence microscopy was used to visualize intracellular transport of newly synthesized HA-Pma1. Wild-type HA-Pma1 after a 2-hour induction (i.e. derepression) is predominantly localized at the plasma membrane (Fig. 1A, top right panel), reflecting its rapid transport through the secretory pathway. After a 2-hour induction at 37°C, HA-Pma1-10 was observed accumulated in ER membranes, coincident with the ER marker, Kar2 (Fig. 1A, middle panels); before induction (0'), no anti-HA staining was observed (top left panel). Methionine was then added to shut off further synthesis; after a 2-hour chase, no signal was detectable, suggesting degradation of HA-Pma1-10 (off, bottom left panel). After induction, in pep4 cells in which vacuolar proteolysis is blocked (Jones, 1991), two regions of staining were observed in some cells, a ring coincident with the ER marker Kar2 (Fig. 1B, arrows), and staining coincident with the lumen of vacuoles, seen by DIC as cellular indentations. After chase in pep4 cells, the ER rings disappeared but vacuolar staining persisted (Fig. 1B). These results suggest that export of newly synthesized HA-Pma1-10 from the ER is slow, after which intracellular transport to the vacuole is rapid. Previously, we established that vacuolar delivery of Pma1-10 occurs via the plasma membrane because Pma1-10 degradation is dependent on the exocyst component Sec6 and the endocytic protein End4 (Gong and Chang, 2001).
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Ubiquitylation of Pma1-10 at or before arrival at the plasma membrane is reversible
The turnover of many plasma membrane proteins is regulated by ubiquitylation, and it has been suggested that ubiquitylation also serves a quality control function to remove defective cell-surface proteins (Hicke and Dunn, 2003; Sharma et al., 2004). Therefore, we tested whether Pma1-10 removal from the plasma membrane is mediated by ubiquitylation. Newly synthesized HA-Pma1-10 was induced in the endocytosis mutant, end3-1, for 2 hours at 37°C, and then immunoprecipitated from cell lysate with anti-HA antibody. Immunoprecipitates were analyzed by western blot with anti-ubiquitin. For these experiments with our end3 strain, induction of HA-Pma1-10 with the MET25 promoter was poor. Therefore, HA-pma1-10 was placed under the control of a MET3 promoter (which, like MET25, is controlled by environmental methionine levels). MET3 is less `leaky' than MET25 such that repression is enhanced in the presence of methionine (data not shown). Very little HA-Pma1-10 is detectable by western blot under repressing conditions (Fig. 2A, 0); substantial induction (derepression) occurred upon removal of methionine (Fig. 2A, on). Methionine was then added back; in end3 cells defective in endocytosis, HA-Pma1-10 is stabilized whereas some HA-Pma1-10 disappears after chase in END3+ cells (Fig. 2A, off).
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). Therefore, we tested whether Rsp5 plays a role in promoting rapid internalization of Pma1-10. Newly synthesized HA-Pma1-10 was analyzed by metabolic pulse-chase experiment in RSP5+ and rsp5-1 cells. HA-Pma1-10 induced in RSP5+ cells is rapidly degraded (Fig. 3A). Interestingly, however, when mutant Pma1-10 is expressed in cells with a chromosomal copy of wild-type Pma1 (right panel), a small fraction of HA-Pma1-10 is protected from degradation, compared with degradation in cells where Pma1-10 is the sole copy of Pma1 (left panel) (notably after 30 and 60 minutes' chase). This protection is probably caused by formation of hetero-oligomers between wild-type and mutant Pma1 (Lee et al., 2002; Wang and Chang, 2002) which stabilize the mutant protein. In rsp5-1 cells at the restrictive temperature, 37°C, there is significant stabilization of HA-Pma1-10 (Fig. 3A, bottom right panel). We then asked whether Rsp5 catalyzes ubiquitin conjugation to Pma1-10 by analyzing ubiquitylation of Pma1-10 in rsp5-1 and RSP5+ cells. After induction for 2 hours at 37°C, HA-Pma1-10 was immunoprecipitated from cell lysate, and immunoprecipitates were analyzed by western blot with anti-ubiquitin. Surprisingly, HA-Pma1-10 ubiquitylation was apparent in rsp5-1 cells at 37°C. These results suggest that stabilization of HA-Pma1-10 is an indirect effect of rsp5-1, perhaps on the endocytic machinery.
Turnover of Pma1-10 from the plasma membrane requires epsin proteins
It is possible that ubiquitylation of Pma1-10 serves as a signal for its internalization from the cell surface. Epsin family proteins and the epsin binding partner, Eps15, are accessory factors that are required at the internalization step in endocytosis, contain ubiquitin-interacting motifs (UIMs) and ubiquitin-associated domains (UBAs), respectively, and have been suggested to play a role in mediating cargo selection (Shih et al., 2002; Wendland, 2002). In yeast, the epsins Ent1 and Ent2 are essential for viability; ent1
ent2 cells bearing temperature-sensitive ent1 on a plasmid are defective in membrane internalization, as revealed by endocytosis of the fluorescent lipophilic dye FM 4-64 (Wendland et al., 1999). Ent1 and Ent2 also have overlapping function with Ede1, the Eps15 homologue (Shih et al., 2002). ent1 ent2 ede1 cells carrying plasmid-borne ent1 lacking its UIM domain (ent1UIM) fail to bind ubiquitin and are unable to internalize the mating pheromone 
factor (Shih et al., 2002). Although the ent1 ent2 ede1 cells bearing ent1UIM have impaired cargo recognition (Shih et al., 2002), they appear to be fully competent to internalize membrane, as revealed by FM4-64 internalization; no delay in vacuolar delivery of FM 4-64 was detectable (Fig. 4A). We then tested whether Pma1-10 removal from the plasma membrane is dependent on these accessory factors. Pulse-chase analysis (Fig. 4B) reveals that HA-Pma1-10 in these cells is stabilized. Fig. 4C shows indirect immunofluorescence localization of HA-Pma1-10 in ent1 ent2 ede1 cells bearing ent1UIM. As in wild-type cells (Fig. 1A, on), newly synthesized HA-Pma1-10 in ent1UIM cells was observed in ER rings (Fig. 4C, on). After chase, however, HA-Pma1-10 is localized to the plasma membrane in ent1UIM cells (Fig. 4C, off), in contrast to ENT1+ cells in which HA-Pma1-10 disappears after chase (Fig. 1C, off, lower left panel). These results suggest that the turnover of Pma1-10 from the plasma membrane requires ubiquitin recognition by the endocytic machinery.
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). After induction, newly synthesized HA-Pma1-10 fails to migrate with detergent-resistant fractions, remaining instead in fractions 4, 5 and 6, like the detergent-soluble marker alkaline phosphatase (Fig. 5A, gradient i); by contrast, preexisting Pma1 prefers to associate with DIGs in fraction 1. These results are in agreement with our previous report that newly synthesized Pma1-10 fails to achieve Triton insolubility, although wild-type Pma1 becomes Triton-insoluble shortly after synthesis (Gong and Chang, 2001).
Remarkably, however, after a 2-hour chase at 37°C in end3-1 cells, HA-Pma1-10 is predominantly distributed with detergent-resistant membranes at the top of the Optiprep gradient (Fig. 5A, ii). In contrast to end3-1, HA-Pma1-10 remains detergent-soluble after chase in a pep4 mutant in which vacuolar degradation is impaired (Fig. 5A, iv). Thus, HA-Pma1-10 is stabilized but remains coincident with the non-raft marker alkaline phosphatase.
Cell fractionation on Renografin density gradients was used to separate plasma membrane from intracellular membranes; plasma membrane remains in the higher density fractions while all other intracellular membranes migrate in the lower density fractions at the top of the gradient (Chang, 2002). Fig. 5B shows that newly synthesized HA-Pma1-10 after chase in end3 cells is accumulated at the plasma membrane, coincident with the cell-surface marker Gas1; this is in agreement with results from indirect immunofluorescence localization (Gong and Chang, 2001). Similarly, stabilization of Pma1-10 at the cell surface in a suppressor strain, yvh1 (discussed below), occurs concomitantly with DIG-association (Fig. 5A, iii).
After arrival and residence at the plasma membrane, wild-type Pma1 achieves its maximally phosphorylated state as revealed by its slow electrophoretic mobility (Chang and Slayman, 1991). Fig. 5C compares phosphorylation of newly synthesized mutant and wild-type Pma1 after a 2-hour chase. In a pep4 background, newly synthesized Pma1-10 has a faster electrophoretic mobility, reflecting its hypophosphorylation (Gong and Chang, 2001). In end4-1 cells, decreased Pma1-10 mobility is consistent with its increased phosphorylation that occurs upon sustained residence at the plasma membrane.
Ubiquitylation does not prevent DIG association
Association of wild-type Pma1 with DIGs is reported to occur shortly after its synthesis, perhaps before ER export (Lee et al., 2002). Because it is possible that the failure of Pma1-10 to associate with DIGs (Gong and Chang, 2001) is dictated by its ubiquitylation, we tested whether ubiquitin can cause exclusion of wild-type Pma1 from detergent-resistant membranes. Monoubiquitin fused to the C-terminus of HA-tagged wild-type Pma1 is delivered to the plasma membrane; Pma1-Ub has increased turnover compared with wild-type Pma1 but is longer lived than Pma1-10 (Shih et al., 2000). DIG association of Pma1-Ub was compared with that of Pma1 by extraction with Triton X-100 followed by centrifugation on Optiprep density gradients. After induction of synthesis for 2 hours (by shifting to galactose-containing medium), HA-tagged Pma1-Ub and Pma1 are comparably associated with DIGs in fractions 1 and 2 of an Optiprep gradient (Fig. 6). These results suggest that ubiquitylation does not prevent DIG association.
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There is no increase in synthesis of Pma1-10 in yvh1 cells (data not shown); rather pulse-chase analysis shows that newly synthesized Pma1-10 is stabilized in yvh1 cells (Fig. 7B). A general impairment of endocytosis in yvh1 cells cannot account for Pma1-10 stabilization (Fig. 7C); degradation of the a-factor receptor, Ste3, which undergoes constitutive vacuolar delivery from the cell surface (Chen and Davis, 2002), is not affected in yvh1 cells. Cell fractionation on Renografin density gradients shows that Pma1-10 is localized at the plasma membrane after 1 hour of chase in yvh1 cells whereas it is localized to intracellular (vacuolar) membranes in pep4 cells (Fig. 7D). Because YVH1 encodes a dual specificity protein phosphatase (Beeser and Cooper, 2000; Guan et al., 1992), we examined the phosphorylation state of Pma1-10 in yvh1 cells. The electrophoretic mobility of Pma1-10 after a 2 hour chase in a yvh1 background is comparable to that of wild-type Pma1, whereas Pma1-10 stabilized by pep4 mutation has increased mobility, indicating hypophosphorylation (Fig. 7E). Consistent with its stability at the cell surface in yvh1 cells, Pma1-10 is found associated predominantly with DIGs (Fig. 5A, iii).
Suppression of Pma1-10 ubiquitylation independent of its slow ER export
Indirect immunofluorescence was used to analyze whether the earliest detectable phenotype of pma1-10 cells, slow ER export, is affected by yvh1 cells. ER localization of newly induced HA-Pma1-10 in yvh1 cells was similar to that seen in YVH1+ cells (Fig. 8A. and refer to Fig. 1A). These results suggest that yvh1 may exert its effect on Pma1-10 behavior after its export from the ER.
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; Harris et al., 1994; Nakamoto et al., 1998). Additional post-ER sites for quality control have been identified and characterized using other Pma1 mutants. A temperature-sensitive Pma1-7 mutant fails to target to the plasma membrane and is routed from the Golgi into the endosomal/vacuolar system for degradation (Chang and Fink, 1995; Luo and Chang, 2000). A Golgi-based quality control mechanism has been reported to mediate disposal of a number of substrates in yeast as well as mammalian cells (Arvan et al., 2002; Trombetta and Parodi, 2003). A post-Golgi or peripheral quality control mechanism has been proposed to account for the rapid turnover of conformationally unstable plasma membrane proteins from the cell surface (Fayadat and Kopito, 2003; Li et al., 1999; Sharma et al., 2004). Similar to our paradigm for quality control of Pma1-10, increased plasma membrane turnover of certain mutant variants of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by ubiquitylation and preferential association of the CFTR mutants with the endocytic machinery (Sharma et al., 2004).
In this study, we report that Pma1-10, which fails to remain stable at the plasma membrane, is ubiquitylated. We propose that ubiquitylation acts as part of a quality control mechanism at the cell surface that monitors the conformational integrity of newly arrived proteins at the plasma membrane (Sharma et al., 2004). Because only a very small fraction of Pma1-10 is ubiquitylated, it is possible that attachment of ubiquitin to one or two monomers is sufficient to promote recognition of the Pma1 oligomer. It has been suggested that Pma1 is a hexamer (Kuhlbrandt et al., 2002) or dodecamer (Lee et al., 2002). Ubiquitin attachment to Pma1-10 may initiate internalization by recruiting epsins and their binding partners, including Ede1 and End3. In support of this model, ent1 ent2 ede1 cells bearing ent1UIM appear fully competent for endocytic delivery of the membrane marker FM 4-64 but are impaired in degradation of newly synthesized Pma1-10 (Fig. 4A). Moreover, indirect immunofluorescence reveals Pma1-10 is stabilized at the plasma membrane in these cells. These results are consistent with in vivo recognition of ubiquitylated substrate by epsins and their binding partners. Nevertheless, we cannot exclude the possibility that loss of the epsin UIM domain has an indirect effect on internalization and degradation of Pma1-10.
It is interesting that Pma1-7 and Pma1-10 escape detection by ER quality control. We have proposed that Pma1-7 is recognized by a Golgi quality control mechanism that diverts newly synthesized Pma1-7 from Golgi into the endosomal/vacuolar system (Luo and Chang, 1997; Pizzirusso and Chang, 2004). Pma1-10 escapes detection by Golgi quality control and moves readily to the plasma membrane. Although we do not at present understand how this happens, one possibility is that distinct conformational parameters are sampled in different compartments of the secretory pathway. Pma1-7 carries two mutations, P434A and G789S (although only the latter promotes temperature-sensitive growth), whereas Pma1-10 has lesions (A165G and V197I) more proximal in the polypeptide chain. It is becoming increasingly clear that the conformational parameter(s) measured by quality control is often distinct from the biological activity of a protein (Sharma et al., 2004; Welch, 2004); both Pma1-7 and Pma1-10 have sufficient catalytic activity to support growth when they are properly targeted to and stabilized at the plasma membrane, respectively. Another possible explanation for escape from detection is that conformational changes are induced during passage through distinct environments in different organelles; a mutant protein may then be able to assume certain requisite conformations but not others.
A major finding in this study is that Pma1-10 ubiquitylation is reversible (Fig. 2B) and Pma1-10 can acquire DIG association and increased phosphorylation when its residence time at the plasma membrane is extended by blocking endocytosis. After chase in end3-1 cells, Pma1-10 becomes DIG-associated, but in pep4 cells, Pma1-10 is DIG-excluded (Fig. 5). In contrast to wild-type Pma1 which associates with detergent-resistant membranes shortly after its synthesis, perhaps before ER export (Bagnat et al., 2001; Lee et al., 2002), Pma1-10 is only able to achieve DIG association at the plasma membrane under certain conditions (Fig. 5). These observations suggest that DIG association is a dynamic process such that exclusion appears reversible.
Conjugation with ubiquitin per se does not appear to promote DIG exclusion because the Pma1-Ub fusion protein is not significantly excluded from detergent resistant membranes (Fig. 6). The Pma1-Ub fusion protein fails to remain stable at the plasma membrane but is longer lived than Pma1-10 (Shih et al., 2000). Previously, we proposed that DIG association promotes cell-surface stability; indeed, wild-type Pma1 is ubiquitylated when DIG integrity is compromised in lcb1-100 cells impaired in sphingolipid synthesis (Wang and Chang, 2002). A model consistent with these observations is that ubiquitylation of Pma1-10 promotes rapid internalization from the plasma membrane and thereby preempts the stabilizing effect of DIG association.
The ubiquitin ligase Rsp5 regulates turnover of many plasma membrane proteins (Dupre et al., 2004; Hicke and Dunn, 2003; Rotin et al., 2000). We show that Pma1-10 is still ubiquitylated in rsp5-1 cells. At present, Rsp5 is the only ubiquitin ligase shown to play a role in ubiquitin conjugation of cell-surface proteins. Nevertheless, our data support a model in which an alternative enzyme catalyzes ubiquitylation of Pma1-10. Stabilization of ubiquitylated Pma1-10 in rsp5-1 cells (Fig. 3A) suggests an indirect effect. This idea is consistent with the suggestion that Rsp5 activity is required for proper functioning of the endocytic machinery (Dunn and Hicke, 2001).
Previously, it was noted that Pma1-10 has instability at the plasma membrane in common with a G381A mutant characterized by Ferreira et al. (Ferreira et al., 2002; Gong and Chang, 2001). By using indirect immunofluorescence to visualize transport of newly synthesized Pma1-10 through the secretory pathway, we now report that Pma1-10 also shares the slow ER export phenotype of the G381A mutant (Fig. 1). The Pma1-10 changes, A165G and V197I, are predicted to reside in the first cytoplasmic loop between transmembrane segments 2 and 3 of Pma1 (Gong and Chang, 2001). Both mutations are required for temperature-sensitive growth of pma1-10 cells, and both changes are required for both the slow ER export phenotype and rapid cell-surface turnover (Y. Liu, unpublished result). At present, it is not clear if there is any relationship between slow exit from the ER and rapid turnover from the plasma membrane. The observation that yvh1 suppresses the latter phenotype and not the former (Fig. 8) suggests that yvh1 acts at a later stage of Pma1-10 quality control.
Work is in progress to determine the molecular mechanism for suppression of pma1-10 by yvh1. The different behavior of Pma1-10 and Pma1-7 in response to yvh1 reflects the sorting differences between the two mutants, the former being removed from the plasma membrane and the latter being removed from the Golgi. Ubiquitylation of Pma1-10, but not Pma1-7, is prevented in yvh1 cells (Fig. 8B). Yvh1 is a dual-specificity protein phosphatase induced by nitrogen starvation (Guan et al., 1992). Cooper and co-workers have suggested that Yvh1 may play a role in the cAMP-dependent protein kinase cascade; yvh1 mutants have phenotypes associated with cAMP-dependent pathways, including growth, glycogen accumulation, and sporulation defects (Beeser and Cooper, 2000). Curiously, Yvh1 participation in these activities has been proposed to be independent of its catalytic activity (Beeser and Cooper, 2000; Muda et al., 1999). Although Pma1-10 has increased phosphorylation in a yvh1 mutant, our preliminary results also indicate that Yvh1 catalytic activity is not required, suggesting that Yvh1 does not directly dephosphorylate Pma1-10 (Y.L., unpublished result). Because yvh1 cells also have impaired stress response element (STRE)-mediated gene expression (Beeser and Cooper, 2000), work is underway to test the intriguing possibility that stress-induced STRE-mediated gene expression participates in maintaining quality control at the plasma membrane.
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200 lys2201 leu2-3,112 ura3-52 ade2); XGY32 and XGX28-1A are pma1-10 and pma1-10 pep4, respectively (Gong and Chang, 2001). A genetic screen was performed by transformation of pma1-10 (XGY32) with a LEU2-marked insertional mutagenic library (Burns et al., 1996). The pma1-10 yvh1::LEU2 strain isolated by genetic screen (KKY11) was backcrossed three times with pma1-10 to generate KKX103-2B; this was then crossed to wild-type (L3852) to generate KKX15-1B (MATa yvh1::LEU2). KKY66 is the original pma1-10 yvh1 isolate transformed with pKK4. KKY1 is L3852 transformed with pKK4. KKY67 is ACY67 (MATa his3200 lys2201 leu2-3,112, ura3-52 ade2 pep4) transformed with pKK4; KKY80 is L3852 transformed with pCC2. end3-1 (RH266-1D; MATa end3-1 leu2 his4 ura3 bar1-1) and end4-1 (RH268-1C; MATa end4-1 leu2 his4 ura3 bar1-1) were obtained from Howard Riezman (University of Geneva, Switzerland). XGX42-8B and 8D are pma1-10 end4-1 and end4-1, respectively; these ascospores come from a cross between XGY32 and RH268-1C. KKY65 is end3-1 transformed with pKK4; KKY81 is end3-1 transformed with pCC2. WLY104 is L3852 transformed with pWL10, a URA3-marked YIp bearing MET25-PMA1 (Luo and Chang, 2000). FY1810 (MATa GAL2+ his4-912 lys2-128 leu21 rsp5-1) and FY354 (MATa his4-912 lys2-128 leu21) are from Fred Winston (Harvard University, Cambridge, MA). LHY3156 and LHY3185, obtained from Linda Hicke (Northwestern University, Evanston, IL), are MATa ede1::KAN ent2::KAN ent1::HIS3 ura3 his3 leu2/trp1 bar1 bearing [pent1-UIM-TRP1] and [p2µ-EDE1-URA3] and [pENT1-TRP], respectively.
Molecular biology
pKK4 is a yeast integrating plasmid (pRS306) (Sikorski and Hieter, 1989) bearing HA-Pma1-10 under the control of the MET25 promoter. pKK4 was constructed by replacing the 3.8 kb ClaI fragment from pXG39 (Gong and Chang, 2001) with that of pWL10 (Luo and Chang, 2000). pKK7 and pKK5 were constructed by placing a 4.5 kb SacI-XhoI fragment from pKK4 into pRS315 and pRS316, respectively. pCC2 is a plasmid bearing HA-Pma1-10 under the control of the MET3 promoter (Mountain et al., 1991). pCC2 was constructed by using primers 234 (5'-CGCGAGCTCTCCAACGATATGTACGTAGTG-3') and 235 (5'-TCCCCCGGGTATCACAACTGTTACGACAGA-3') to amplify a 500 bp SacI-SmaI fragment using PCR; this fragment was swapped with that from pKK4. pKK42, a LEU2-marked centromeric plasmid bearing HA-Pma1-10 under the control of the MET3 promoter, was constructed by replacing the 0.5 kb SacI-XmaI fragment of pKK7 with that from pCC2. pS3 is a HIS3-marked centromeric plasmid bearing HA-Pma1-7 under the control of the MET25 promoter (Pizzirusso and Chang, 2004). pXZ28 (obtained from Jim Haber, Brandeis University, Waltham, MA) and pLH609 (obtained from Linda Hicke) are URA3-marked centromeric plasmids bearing wild-type Pma1 and Pma1 with K48R ubiquitin fused in-frame at the C-terminus, respectively; both inserts are under the control of the GAL1 promoter. pSL2015, provided by Nick Davis (Wayne State University, Detroit, MI) is a URA3-marked centromeric plasmid bearing myc-tagged STE3 under the control of the GAL1 promoter.
Protein induction, western blot and endocytosis assay
Cells with plasmids bearing wild-type or mutant tagged Pma1 under the control of the MET25 or MET3 promoters were induced in the same way (Luo and Chang, 2000). Cells were grown overnight at 25°C in medium containing 600 µM methionine; mid-log cultures were washed once with water and then resuspended in medium without methionine and shifted to 37°C. To assess protein induction, lysates were assayed by western blotting before methionine removal (time 0), after 1-2 hours without methionine (`on'), and after adding back 2 mM methionine (`off'). For induction of genes under GAL1 control, cells were grown to mid-log phase at 30°C in synthetic complete medium containing 2% raffinose, and then transferred to medium containing 2% galactose for 2 hours. For western blotting, lysate was prepared by vortexing cells with glass beads in the presence of a protease inhibitor cocktail, as previously described. Protein levels were normalized (Bradford assay). Western blots with anti-ubiquitin antibody (Zymed Laboratories) were processed after autoclaving nitrocellulose filters, as described previously (Swerdlow et al., 1986).
Endocytosis of Ste3 was assayed as described previously (Luo and Chang, 2000). Cells bearing pGAL-myc-STE3 were grown overnight in synthetic complete medium without uracil and 2% galactose. The internalization assay was initiated by adding 3% glucose to mid-log cultures. Aliquots were removed at various times for lysis and western blot with anti-myc antibody. To assay endocytosis of FM 4-64, cells were grown in YPD to mid-log, resuspended at 20 OD600/ml and incubated with 40 µM FM 4-64 at 30°C for 15 minutes. Cells were pelleted and resuspended at 5 OD600/ml for 45 minutes at 30°C before visualization.
Metabolic labeling, immunoprecipitation and cell fractionation
For pulse-chase experiments, cells were grown to mid-log phase in supplemented minimal medium. Cells were harvested and resuspended in fresh medium before metabolic labeling with Expre35S35S, and chasing for various times after adding an equal volume of synthetic complete medium containing 20 mM cysteine and methionine. Immunoprecipitates were with anti-Pma1 polyclonal antibody and normalized to acid-precipitable cpm, and analyzed by SDS-PAGE and fluorography.
In order to exclude the possibility of co-immunoprecipitation, immunoprecipitates for western blot with anti-ubiquitin were performed by adding 1% SDS to lysate (usually 200 µg protein) before diluting into RIPA buffer without SDS to a final SDS concentration of 0.1% SDS. Monoclonal anti-HA antibody (Covance) was then added.
For cell fractionation, lysate was fractionated on Renografin density gradients as described previously. Renocal-60 (60%) was substituted for Renografin-76. After centrifugation of density gradients, fourteen fractions were collected from the top and membranes were pelleted by centrifugation at 100,000 g for 1 hour before analysis by SDS-PAGE and western blot.
Indirect immunofluorescence
Cells stained with monoclonal anti-HA and fluorescent secondary antibodies (Jackson Immunochemicals) were visualized with an Olympus fluorescence microscope and images were collected with a Hamamatsu Orca CCD camera. Anti-Kar2 polyclonal antibody was a gift from Mark Rose (Princeton University, Princeton, NJ).
Association with detergent-resistant membranes
Detergent-insoluble glycolipid (DIG)-enriched complexes were isolated as described previously (Bagnat et al., 2000). Lysate (50 µg protein) was incubated with 1% cold Triton X-100 in 100 µl for 30 minutes on ice, and then mixed with Optiprep and placed at the bottom of a centrifuge tube. The sample was overlayed with an Optiprep step gradient and centrifuged in a Beckman TLS 55 rotor at 200,000 g for 2 hours. Six fractions were collected from the top; proteins were acid-precipitated for analysis by SDS-PAGE and western blot.
Genetic selection
To isolate suppressors of pma1-10, mutations were introduced in XGY32 cells by transformation with a LEU2- and lacZ-marked insertional library (Burns et al., 1996). Approximately 35,000 transformants were plated at 37°C. Colonies that grew at 37°C were backcrossed to pma1-10 cells and the diploid was subjected to tetrad analysis to identify single LEU2-marked insertions that suppressed temperature-sensitive growth. A secondary screen was performed by western blot to identify suppressors that had an increased level of Pma1-10 protein by comparison to XGY32 cells after shifting to 37°C for 6 hours. Sequence analysis of genomic DNA adjacent to the insertion was performed as described (Burns et al., 1996). yvh1 is one of three suppressors isolated.
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References |
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