Involvement of Specific COPI Subunits in Protein Sorting from the Late Endosome to the Vacuole in Yeast^▿

Plasmids.

Plasmids generated for this study are listed in Table ​Table2.2. Plasmids pGFP-CPS1, pGFP-VPS27, and pSTE2-GFP which express GFP-CPS1 (41), GFP-VPS27 (35), and STE2-GFP, respectively, were generously provided by S. Emr (University of California, San Diego). Plasmid pSTE3-GFP, which expresses STE3-GFP, was kindly provided by R. Piper (University of Iowa, Iowa City). Plasmids pRS314STE2-GFP and pRS314STE2Δtail-GFP, which express STE2-GFP or STE2Δtail-GFP (57), respectively, were generously provided by K. Blumer (Washington University School of Medicine, St. Louis, MO). Plasmid pGALΔBglII-CPY[1-50]GFP, which expresses CPY^1-50-GFP, was kindly provided by O. Deloche (Universite de Lausanne, Lausanne, Switzerland).

Fluorescence microscopy.

Yeast cells expressing GFP/RFP fusion proteins were grown to mid-log phase at 26°C and examined by confocal microscopy. For induction of proteins from a GAL promoter (i.e., CPY-GFP), cells were grown in glucose-containing media to mid-log phase and then shifted galactose-containing media until GFP expression was observed.

FM4-64 labeling and visualization.

Yeast cells grown to mid-log phase were stained with the lipophilic dye FM4-64 (Molecular Probes, Eugene, OR), basically as described previously (58). For endosomal staining, cells were incubated with 16 μM of FM4-64 on ice in the dark for 45 min. Afterwards, cells were washed twice with fresh medium and kept on ice prior to observation by confocal microscopy. To induce endocytosis and label the endosomal compartments, cells were transferred to 26°C for 10 min. For vacuolar staining, cells were pulsed with 32 μM of FM4-64 for 20 min in the dark at 26°C. Following the pulse, two chases of 20 min each in medium lacking FM4-64 at 26°C were performed. Labeled cells were observed by confocal microscopy.

Immunoblot assay for CPY and Kar2 secretion.

Yeast cells were either plated or spotted (as 10-fold serial dilutions of cells) on YPD plates at 26°C and grown for 24 h prior to replica plating onto nitrocellulose filters (BA-S85; Schleicher and Schuell). The filters were then placed yeast side up, incubated on a fresh YPD plate, and grown for an additional day. The immunoblotting assay for CPY and Kar2 secretion was performed using standard procedures. Cells were removed by three washes of the membranes for 5 min each with phosphate-buffered saline. No cell lysis was observed on either plates or filters using this technique (data not shown). Filters were then blocked in 5% nonfat dry-milk in 0.1% Tween 20-phosphate-buffered saline for 1 h. After blocking, membranes were probed with polyclonal anti-CPY antibodies (1:1,000; gift of S. Emr, University of California, San Diego) or polyclonal anti-Kar2 antibodies (1:2,000; gift of C. Barlowe, Dartmouth University, Hanover, NH), and detected by ECL enhanced chemiluminescence, using anti-rabbit peroxidase-conjugated antibodies (1:10,000; Amersham Biosciences).

Separation of extracellular and intracellular forms of CPY.

Five optical density at 600 nm (OD₆₀₀) units of yeast grown to mid-log phase was incubated in 500 μl of YPD medium containing 50 mM KPO₄, pH 5.7, for 1 h at 30°C. Then 5 μl of 1 M NaN₃ was added, and the cell cultures were cooled on ice for 10 min. Culture samples were centrifuged to separate the cells (containing the intracellular fraction; I) from the medium (containing the extracellular fraction; E). Cell pellets were resuspended in 150 μl of spheroplast-forming buffer (50 mM Tris-HCl, pH 7.4, 1.4 M sorbitol, 2 mM MgCl₂, 10 mM NaN₃, freshly added 40 mM β-mercaptoethanol, 0.15 mg/ml of Zymolase) and incubated by gentle shaking for 30 min at 30°C. Spheroplasts were lysed by the addition of 50 μl of 2% sodium dodecyl sulfate (SDS) and boiled for 5 min. The lysate was then centrifuged for 10 min at 20,800 × g, and the supernatant was removed for electrophoretic separation on acrylamide gels by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples taken for electrophoresis consisted of an aliquot of 0.1 OD₆₀₀ U for the intracellular fraction and an aliquot corresponding to the volume of 0.4 OD₆₀₀ U in culture (40 μl). SDS sample buffer was added to each sample, and the samples were separated on 8% SDS-PAGE gels. Following transfer to the nitrocellulose membranes, the blots were incubated with polyclonal anti-CPY antibodies (1:1,000; gift of S. Emr, University of California, San Diego) and proteins were visualized using ECL chemiluminescence.

Vacuole-targeted transmembrane proteins accumulate on endosomes adjacent to the vacuole in certain COPI mutants.

To further investigate whether vacuolar protein sorting is generally affected in COPI mutants, we applied a microscopy approach and examined the localization of a number of proteins that undergo trafficking to the vacuole (Fig. ​(Fig.22 and ​and3).3). We first examined the localization of transmembrane proteins known to target to the vacuole (Fig. ​(Fig.22 and ​and3A).3A). These proteins (i.e., CPS1, Fur4, Ste2, and Ste3) reach the vacuolar lumen by means of the MVB internalization and vacuolar fusion machinery in wild-type cells, but may accumulate on the endosomal compartment adjacent to the vacuole in class E vps mutants (10, 34, 41, 55). Proteins which do not undergo internalization at the limiting membrane of MVB are delivered to the limiting membrane of the vacuole, where they remain stabilized (33).

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FIG. 2.

Vacuole-targeted transmembrane proteins accumulate on endosomes and do not reach the vacuolar lumen in certain COPI mutants. (A) Fur4 does not target to the vacuolar lumen in COPIb mutants. Wild-type (WT) yeast (W303-1a) and strains with mutations in COPIf (sec21-1, sec21-2, and ret2-1), COPIb (sec27-1, sec28Δ [RDY241], sec33-1, and ret1-3), ESCRT (vps23Δ), and endocytosis (end4-1) were transformed with a multicopy plasmid expressing FUR4-GFP (pADH-FUR4-GFP) and examined by confocal microscopy. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated. (B) Ste2 does not target to the vacuolar lumen in a COPIb mutant. Wild-type yeast (W303-1a) and a COPIb mutant (sec28Δ [RDY241]) were transformed with a single-copy plasmid producing either Ste2-GFP (pRS314-STE2-GFP) or Ste2Δtail-GFP (pRS314-STE2Δtail-GFP; Ste2Δtail). Strains with mutations in COPIf (sec21-1, sec21-2, ret2-1, and ret3-1), COPIb (sec33-1 and ret1-3), ESCRT (vps23Δ), and END4 (end4-1) were transformed with a multicopy plasmid expressing STE2-GFP (pSTE2-GFP). Arrows indicate the position of endosomal compartments adjacent to the vacuole in which GFP-tagged markers accumulate. (C) Ste3 does not target to the vacuolar lumen in COPIb mutants. Wild-type yeast (W303-1a) and strains with mutations in COPIf (sec21-1, sec21-2, ret2-1, and ret3-1), COPIb (sec27-1, sec28Δ [RDY241], sec33-1, and ret1-3), ESCRT (vps23Δ), and END4 (end4-1) were transformed with a single-copy plasmid expressing STE3-GFP (pSTE3-GFP) and examined by confocal microscopy. Arrows indicate the position of endosomal compartments adjacent to the vacuole in which Ste3-GFP accumulates.

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FIG. 3.

CPS1 localization is impaired, although CPY^1-50GFP localizes normally, in certain COPI mutants. (A) CPS1 does not target to the vacuolar lumen in COPIb mutants. Wild-type (WT) yeast (W303-1a) and strains with mutations in COPIf (sec21-2, ret2-1, and ret3-1), COPIb (sec27-1, sec28Δ [RDY241], sec33-1, and ret1-3), ESCRT (vps23Δ), and RCY1 (rcy1Δ) were transformed with a multicopy plasmid expressing GFP-CPS1 (pGFP-CPS1) and examined by confocal microscopy. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated. (B) CPY-GFP localizes to the vacuole in COPIb mutants. Wild-type yeast (W303-1a) and strains with mutations in COPIb (sec27-1 and sec28Δ [RDY241]), COPIf (sec21-1, ret2-1, and ret3-1), ESCRT (vps23Δ), and RCY1 (rcy1Δ) were transformed with a single-copy plasmid expressing CPY^1-50-GFP (pGALΔBglII-CPY[1-50]GFP) and examined by confocal microscopy.

We first examined delivery of the lumenal vacuolar resident enzyme, CPS1, by monitoring localization of a GFP-tagged form of CPS1 (34, 41). We found that like an ESCRT-I mutant (vps23Δ), GFP-CPS1 is not delivered to the vacuolar lumen in COPIb mutants (Fig. ​(Fig.3A).3A). Notably, in sec27-1 and sec28Δ cells (those used in Fig. ​Fig.1A),1A), GFP-CPS1 was present on the limiting membrane of the vacuole and in large punctate structures located adjacent to the vacuole. This contrasts with wild-type cells or rcy1Δ cells, which are deficient in early endosome-Golgi protein recycling (24), wherein GFP-CPS1 labeling is present in the vacuolar lumen (Fig. ​(Fig.3A).3A). Likewise, GFP-CPS1 localized to the vacuolar lumen in COPIf mutants (e.g., sec21-1, ret2-1, and ret3-1) (Fig. ​(Fig.3A),3A), suggesting that they have no effect upon CPS1 sorting to MVB pathway. Finally, we examined two alleles of αCOP (sec33-1 and ret1-3), a COPIb component, and found that GFP-CPS1 sorting was partially affected in sec33-1 cells, but was not affected in ret1-3 cells. Together, these results indicate that only mutations in COPIb subunits have an effect upon CPS1 sorting, but they may be allele specific.

Next, we examined the localization of GFP-tagged Fur4, a protein that resides on the plasma membrane, but is degraded in the vacuole through the MVB pathway (34) or recycled through a late endosome back to the cell surface (10). As expected, Fur4-GFP yields labeling of the vacuolar lumen in wild-type cells, but localizes to the plasma membrane in an endocytosis-defective mutant, end4-1 (49) (Fig. ​(Fig.2A).2A). In contrast, Fur4-GFP is present on the plasma membrane and accumulates in intracellular compartments in specific COPIb mutants (sec27-1 and sec28Δ), as well as the vps23Δ (Fig. ​(Fig.2A)2A) and vps37Δ (data not shown) ESCRT-I mutants (Fig. ​(Fig.2A).2A). A partial defect in Fur4-GFP delivery to the vacuole was observed in sec21-1 cells, but was not observed in the sec21-2 COPIf and ret1-3 COPIb mutants (Fig. ​(Fig.2A2A and Table ​Table3),3), which show defects in anterograde ER-to-Golgi apparatus transport at 26°C.

We then examined the localization of GFP-tagged versions of Ste2 and Ste3, which are mating factor receptors that undergo downregulation through internalization from the plasma membrane and delivery to the vacuole by sorting to the MVB vesicles (34). We found that Ste2-GFP expressed from a single-copy plasmid labeled the vacuole and plasma membrane in wild-type cells, but was mainly concentrated in a large punctate structure adjacent to the vacuole in the sec28Δ mutant (RDY241; Fig. ​Fig.2B).2B). In contrast, sec28Δ cells expressing a truncated version of Ste2-GFP, which does not undergo internalization from the cell surface (Ste2Δtail) (57), yielded typical plasma membrane labeling. This demonstrates that Ste2-GFP requires internalization to accumulate at a compartment adjacent to the vacuole in the COPIb mutant. Ste2-GFP expressed from a multicopy plasmid in sec33-1 cells was partially impaired in its delivery to the vacuole. In contrast, in sec21-1, sec21-2, ret2-1, ret3-1, and ret1-3 cells Ste2-GFP trafficked properly. When expressed from a single-copy plasmid, Ste3-GFP was unable to reach the vacuole in either sec28Δ (RDY241) or sec27-1 mutants (Fig. ​(Fig.2C),2C), but was trafficked properly in sec33-1 and sec21-2 cells. In end4-1 cells, which are defective in endocytosis (49), both Ste2-GFP (Fig. ​(Fig.2B)2B) and Ste3-GFP (Fig. ​(Fig.2C)2C) labeled the plasma membrane but were unable to be internalized, as expected. Finally, in the control class E vps mutant (vps23Δ), both Ste2-GFP (Fig. ​(Fig.2B)2B) and Ste3-GFP (Fig. ​(Fig.2C)2C) accumulated at compartments adjacent to the vacuole, similar to what was observed in the sec28Δ and sec27-1 COPIb mutants.

Together, these results imply that specific mutations in COPIb subunits missort transmembrane proteins to either the limiting membrane of the vacuole or a large late endosomal compartment adjacent to the vacuole, in the case of CPS1 (Fig. ​(Fig.3A),3A), or endosomal compartments adjacent to the vacuole, in the case of recycling plasma membrane proteins, Fur4 (Fig. ​(Fig.2A),2A), Ste2 (Fig. ​(Fig.2B),2B), and Ste3 (Fig. ​(Fig.2C).2C). While our immunoblot assay (Fig. 1A, B, and E) indicated that endosomal transport is affected in some COPI mutants, the mislocalization of various vacuolar markers clearly demonstrates that a late step in vacuolar protein sorting is impaired therein.

The CPY-GFP soluble vacuolar hydrolase localizes normally in COPI mutants.

The fact that vacuole-targeted transmembrane proteins are mislocalized in ESCRT-I and certain COPIb mutants suggests that COPIb may participate in the process of MVB protein sorting and/or biogenesis. To examine this further, we determined the localization of a soluble endocytic cargo whose transport does not depend on the MVB internalization, namely CPY. Although CPY is secreted in part from certain COPIb mutants (Fig. ​(Fig.11 and Table ​Table3),3), we examined whether an intracellular fraction of this soluble vacuolar marker reaches the vacuole. Since CPY delivery to the vacuole does not require internalization into the MVB (50), unlike CPS1, it has a possibility of reaching the vacuolar lumen due to accumulation of m-CPY even in the intracellular fraction of class E vps mutants (2, 45). We examined CPY delivery by monitoring the localization of a GFP-tagged and truncated form of CPY (CPY^1-50-GFP) (17) in COPIb and other mutants (Fig. ​(Fig.3B).3B). We found that CPY^1-50GFP labeled the vacuolar lumen in both sec27-1 and sec28Δ (RDY241) cells, as well as in COPIf (sec21-1, ret2-1, and ret3-1) mutants, an ESCRT-I mutant (e.g., vps23Δ), and rcy1Δ cells. Thus, while a portion of CPY is secreted from COPIb mutants (Fig. 1A, B, C, and E), another portion is delivered properly. These findings are consistent with previous observations showing that CPY processing is normal in both sec27-1 and sec28Δ cells at permissive temperatures (20, 38). The delivery of soluble vacuolar proteins to the vacuole in COPIb mutants, in contrast to the accumulation of transmembrane proteins on the class E compartment therein, further supports the idea of COPIb involvement in the MVB sorting pathway.

Certain COPIb mutants have enlarged late endosomal compartments.

We next examined whether the COPIb mutants accumulate an enlarged late endosome (class E compartment) similar to the established class E vps mutants (2, 3, 50). To determine this, we performed vital staining of vacuoles and endosomes with the lipophilic styryl dye FM4-64 (58). Addition of FM4-64 to yeast leads to dye incorporation into the plasma membrane, which is subsequently endocytosed and transported via endosomal transport intermediates to the vacuole. We examined endosomal staining by pulse-labeling with FM4-64 on ice and a short chase at 26°C, as well as vacuolar labeling by pulse-chase incubation at 26°C (Fig. ​(Fig.4A).4A). We found that in wild-type cells, FM4-64 labeled multiple small intracellular compartments (Fig. ​(Fig.4A,4A, left panels), which correspond to endosomes, after pulse-labeling on ice and the chase at 26°C, as previously described (58). In contrast, certain COPIb mutants (e.g., sec27-1 and sec28Δ [RDY241]) tended to have single large punctate structures located adjacent to the vacuole (Fig. ​(Fig.4A).4A). A similar type of labeling was observed in control vps23Δ cells (Fig. ​(Fig.4A),4A), which are known to have enlarged late endosomes (vps class E compartment) (4). In pulse-chase experiments performed at 26°C, vacuolar labeling was observed in wild-type cells, as well as in the COPIb and vps23Δ mutants (Fig. ​(Fig.4A,4A, right panels). However, single large punctate structures (i.e., late endosomes) were still observed adjacent to the vacuole in the sec27-1, sec28Δ (RDY241), and vps23Δ cells after the chase period. No significant defects in FM4-64 endosomal and vacuolar labeling were obvious in either sec33-1 or the sec21-2 mutants. Thus, altered FM4-64 labeling is exhibited in specific COPIb mutants and the large labeled structure located adjacent to the vacuole closely resembles the class E compartment seen in certain vps mutants (50).

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FIG. 4.

FM4-64 labeling of an enlarged late endosome in certain COPI mutants. (A) FM4-64 staining of endocytic membranes in COPIb mutants. Wild-type (WT) cells (W303-1a) and mutants in COPIf (sec21-2), COPIb (sec27-1, sec28Δ, and sec33-1), and ESCRT (vps23Δ) were grown to mid-log phase and subjected to endosomal and vacuolar labeling with FM4-64 to visualize these organelles (see Materials and Methods). Arrows indicate positions of class E compartment in which FM4-64 accumulates. Phase-contrast microscopy (PC) panels are indicated. (B) Colocalization of FM4-64 with accumulated transmembrane proteins in COPIb mutants. sec28Δ (RDY241) and sec27-1 yeast cells expressing GFP-CPS1 or Fur4-GFP proteins were stained with FM4-64 by pulse-chase labeling at 26°C. Both phase-contrast microscopy (PC) and merged (MERGE) panels are indicated.

Next, we labeled COPIb cells expressing either GFP-CPS1 or Fur4-GFP with FM4-64, by pulse-chase labeling at 26°C (Fig. ​(Fig.4B).4B). This allowed us to determine whether FM4-64 accesses the same enlarged late endosomal compartment in which Fur4 and CPS1 accumulate (Fig. ​(Fig.2A2A and ​and3A,3A, respectively). As expected, we found both GFP-CPS1 and Fur4-GFP colocalized with FM4-64 in an enlarged compartment adjacent to the vacuole in both sec27-1 and sec28Δ cells (Fig. ​(Fig.4B).4B). This colocalization was observed in all cells that had an obvious class E phenotype. This demonstrates that the loss of COPIb function leads to the accumulation of vacuole-bound cargoes in the enlarged late endosome. This effect is not likely to be indirect (i.e., due to defects in retrograde ER-Golgi apparatus transport) since the protein markers examined in this study reached the endosomal compartments and do not accumulate at early compartments (e.g., ER or Golgi apparatus) within the cells. However, we cannot rule out the possibility that a COPI-mediated endosomal recycling event might also alter protein sorting into vesicles.

Vps27 coimmunoprecipitates with COPIb subunits.

The fact that COPIb and class E vps mutants have a similar phenotype suggested that COPIb may be involved directly in the MVB sorting pathway. To address this, we examined whether COPIb subunits are able to bind to Vps27, a protein of the late endosome/MVB that is involved in early steps of MVB biogenesis. We performed co-IP experiments between myc-tagged subunits of COPIb (e.g., myc-Sec27, myc-Sec28, and myc-Sec33) and GFP-Vps27, using anti-myc antibodies (35) (Fig. ​(Fig.5A).5A). These myc-tagged constructs were functional, given their ability to rescue their respective temperature-sensitive mutations (data not shown). In addition, we used a vps23Δ mutant as the strain background as a block in cargo internalization presumably locks the recruited components of MVB machinery onto the endosomal membrane (2, 3, 8, 33).

As shown in Fig. ​Fig.5A,5A, all three tagged COPIb subunits were expressed well and had mobilities on SDS-PAGE gels corresponding to their expected molecular weights, although some degradation products were observed in the lysates of myc-Sec27- and myc-Sec33-producing cells. The latter are probably due to proteolysis during sample preparation. Importantly, GFP-Vps27 coimmunoprecipitated with all three COPIb subunits (Sec27, Sec28, and Sec33), but was not evident in precipitates obtained from cells expressing vector alone. This interaction was also observed in wild-type cells (Fig. 5B and C), indicating the ability of Vps27 to bind to COPIb under normal conditions. Based upon our coimmunoprecipitation data, we estimated by densitometry that 2 to 3% of cellular Vps27 was precipitated by the COPI subunits. Such a low level of association between these COPI components and Vps27 may indicate either a weak interaction between these proteins or, alternatively, that only a small portion of COPI is involved in vacuolar protein sorting. It is also possible that the interaction between COPI components and Vps27 may not be direct, but is mediated by another protein. A nonrelevant myc-tagged protein, Vsm1, did not precipitate Vps27 (Fig. ​(Fig.5C),5C), indicating that COPI subunits precipitate Vps27 specifically. We note that overexpressed GFP-Vps27, but not native Vps27, was observed in these immunoprecipitation experiments, which could be due to low sensitivity of the anti-Vps27 antiserum. In addition, we note that COPI protein-protein interactions are apparently maintained in these cells, as myc-Sec27 and myc-Sec28 were able to coimmunoprecipitate both native and myc-Sec33 protein. To begin characterizing the molecular requirements for the COPI-Vps27 interaction, we examined whether Sec33 could bind Vps27 in cells lacking a nonessential COPI component (sec28Δ cells). As can be seen in Fig. ​Fig.5B,5B, GFP-Vps27 could be precipitated via myc-Sec33 in sec28Δ cells as efficiently as in wild-type and vps23Δ cells, indicating that Sec28 is not essential for their interaction. We also examined whether a COPIf subunit, Sec21, is able to precipitate Vps27, but we could not observe the interaction (Fig. ​(Fig.5B).5B). As the levels of myc-Sec21 were somewhat lower than those of the COPIb subunits, this may partly account for our inability to detect an interaction between Sec21 and Vps27.

To examine Vps27-COPIb complex formation at endogenous levels of protein expression, we integrated a myc tag at the 3′ end of VPS27 and a GFP tag at the 3′ end of SEC28 in wild-type cells. As shown in Fig. ​Fig.5D,5D, Vps27-myc was able to specifically precipitate Sec28-GFP when both were expressed from their native promoters. Vps27 binding to COPIB subcomplex components further supports our hypothesis of COPI involvement in the endosomal protein sorting pathway.

We are grateful to Scott Emr for helpful discussion. We thank Charles Barlowe, Kendall Blumer, Rainer Duden, Scott Emr, Olivier Deloche, Uttam Surana, Robert Piper, Mark Longtine, Andreas Mayer, Peter Novick, Howard Riezman, and Anne Spang for the generous gifts of reagents.

This work was supported by grants to J.E.G. by the Minna James Heineman and Minerva Foundations, Germany. J.E.G. holds the Henry Kaplan chair in Cancer Research.