Correspondence to: Todd R. Graham, Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235. Tel:(615) 343-1835 Fax:(615) 343-6707 E-mail:tr.graham{at}vanderbilt.edu.
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Abstract |
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ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1 synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2
mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro
-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2
cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.
Key Words: adenosine diphosphateribosylation factor, aminophospholipid translocase, clathrin, DRS2, trans-Golgi network
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Introduction |
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THE directed movement of proteins between compartments of the secretory and endocytic pathways is driven by the formation of small coated transport vesicles that bud from a donor compartment, and then fuse with an acceptor compartment. COPI, COPII, and clathrin are the best characterized coat proteins involved in protein transport. COPI, a heptameric (, ß, ß',
,
,
,
-COP) protein complex, coats vesicles that mediate protein transport from the Golgi to the ER and possibly between Golgi compartments. To initiate budding of COPI-coated vesicles from Golgi membranes, the small GTP-binding protein ADP-ribosylation factor (ARF)1 mediates the recruitment of COPI coats to primarily cis Golgi cisternae. ARF is also required to recruit clathrin/AP-1 coats to the trans-Golgi network. Clathrin coats are assembled from triskelions composed of three clathrin heavy chains and three light chains. The tetrameric adaptor protein (AP) complex, AP-1, links membrane proteins to clathrin at the TGN and appears to facilitate cargo delivery to endosomal compartments (reviewed in
In yeast Saccharomyces cerevisiae, ARF is encoded by two nearly identical genes, ARF1 and ARF2, that appear to be functionally redundant. Deletion of both ARF genes is lethal, but strains harboring either an arf1 or an arf2
allele are viable and grow nearly as well as an isogenic wild-type strain (
To gain a better understanding of the essential role that ARF plays in vivo, we have used a genetic screen to identify seven genes (SWA1SWA7) for which mutant alleles exhibit synthetic lethality with arf1. This genetic interaction often predicts that the two gene products function in the same pathway or in parallel pathways. swa5-1 was previously identified as a new temperature-sensitive (ts) allele of the clathrin heavy chain gene (chc1-5), providing support for a functional interaction in vivo between clathrin and ARF (
, if viable, exhibit slow growth, decreased rates of endocytosis, and a defect in retention of late Golgi membrane proteins (
, apm1
, or apl2
with chc1-ts exacerbates defects in growth and retention of Golgi resident proteins relative to the chc1-ts mutant (
Here we report that SWA3 is allelic to DRS2, which encodes an integral membrane P-type ATPase ( mutant has been reported to exhibit a defect in translocation of a fluorescent PS derivative across the plasma membrane (
In addition to the synthetic lethal interaction with arf1, we found that drs2
also exhibits a specific synthetic lethal interaction with chc1-ts alleles. Moreover, the drs2
mutant has a defect in late Golgi function and exhibits several phenotypes at the nonpermissive temperature that suggest a loss of clathrin function at the yeast Golgi in vivo. Consistent with these observations, immunofluorescence and subcellular fractionation studies indicate that Drs2p localizes to late Golgi membranes. These results argue that the primary site of Drs2p function is in the Golgi complex rather than the plasma membrane, and suggest a link between Drs2p and the ARF-dependent recruitment of clathrin to Golgi membranes.
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Materials and Methods |
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Strains and Media
Yeast cells were grown in standard rich medium (YPD), sporulation, or SD minimal media containing required supplements and 0.2% yeast extract where indicated ( leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801 suc2-
9;
(SEY6210 drs2
::TRP1), 6210 arf1
(SEY6210 arf1
::HIS3;
200 trp1-
901 ade 2-101 suc2-
9 drs2
::TRP1), GPY1103 (MATa leu2-3,112 ura3-52 his4-519 trp1 can1 chc1-
8::LEU2;
200 trp1-
901 suc2-
9 ret1-1;
leu2-3,112 ura3-52 trp1 suc2 clc1
::HIS3;
200 trp1-
901 lys2-801 suc2-
9 sec21-1;
200 trp1-
901 sec23-1), TGY144 (MAT
leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801 sec1-1), TGY1906 (MATa leu2-3,112 ura3-52 his3-
200 trp1-
901 suc2-
9 pan1-20), TGY1912 (MATa leu2-3,112 ura3-52 his3-
200 trp1-
901 lys2-801 suc2-
9 end4-1), SEY5185 (MATa leu2-3,112 ura3-52 sec18-1), BHY161 (SEY6210 vps21
::HIS3), BHY163 (SEY6210 ypt7
::HIS3), 6210 vps33
(SEY6210 vps33
::HIS3), and 6210 vps35
(SEY6210 vps35
::HIS3).
Cloning of SWA3 and Plasmid Construction
To clone SWA3, strain CCY2808 (swa3-2;
pPR10 contained a fragment of chromosome I from coordinates 94916104338. Deletion of the BamHI fragment contained solely in DRS2 (to produce pPR10BamHI) destroyed the complementing activity of pPR10. The full-length DRS2 gene contained on a SpeI-SnaBI fragment was subcloned into SpeI-SnaBI digested pRS315 or pRS425 to produce pRS315-DRS2 and pRS425-DRS2. Both plasmids complemented the cold-sensitive growth defect of CCY2808. A drs2 deletion plasmid (pGCR1) was constructed by replacing a BamHI-SnaBI fragment in pPR10 with an ~1.1-kb BamHI-PvuII fragment containing TRP1 from pJJ280 (
. The correct integration event was confirmed by PCR.
Site-directed mutagenesis of DRS2 was performed by the megaprimer PCR method (
The 2-µm KEX2HA plasmid was constructed by subcloning a SalI/EagI digestion fragment of pSN218 (
Cell Labeling and Ste3p Turnover
Cell labeling, immunoprecipitation (
Subcellular Fractionation and Preparation of Clathrin-coated Vesicles
Subcellular fractionation was performed as previously described ( as previously described (
Electron Microscopy
Spheroplasts were prepared for electron microscopy as previously described (
Preparation of Antiserum Against Drs2p
A TrpE-DRS2 fusion construct was prepared by ligating the BglII/HindIII cut DRS2 fragment (1,180 bp) from pPR10 into the BamHI/HindIII-digested pATH2 vector (
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Results |
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drs2 Is Synthetically Lethal with arf1
and chc1-ts Alleles
To clone the SWA3 gene, a yeast genomic library (CEN, LEU2) was used to transform the swa3-1 mutant, and the transformants were screened for complementation of the cold-sensitive growth and arf1 synthetic lethal phenotypes. A single library plasmid was isolated that was able to complement both mutant phenotypes and further subcloning analyses revealed that a 5-kb SpeI-SnaBI genomic fragment containing the full-length DRS2 gene (and no other open reading frame, ORF) retained the complementing activity (see Materials and Methods).
A drs2 null strain was generated by replacing most of the DRS2 coding sequences with TRP1. Consistent with the phenotype previously reported for drs2 strains (
null mutant was unable to grow at 20°C or below, but grew well at temperatures above 23°C. Linkage analysis indicated that SWA3 is allelic to DRS2, and synthetic lethality between arf1
and drs2
was confirmed by crossing the single mutants and characterizing the progeny by tetrad analysis (Figure 1 A and data not shown). This genetic interaction suggested that Drs2p may be involved in an ARF-dependent vesicle-mediated protein transport event(s).
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To determine if drs2 would exhibit synthetic lethality with other mutations that perturb transport vesicle formation, we crossed drs2
with strains harboring ts mutations in subunits of COPI (ret1-1, sec21-1, and sec27-1 encoding
,
, ß'-COP, respectively), the ER transport vesicle coat COPII (sec12-1 and sec23-1), and the clathrin heavy chain (chc1-5, chc1-
57, chc1-521), and analyzed the progeny by tetrad dissection. All of the sec drs2
and the ret1 drs2
double mutants were obtained at the expected frequency (approximately one fourth of the progeny) and most grew well at 26.5°C (Figure 1 A). The exception was sec21-1 drs2
mutants, which grew somewhat more slowly at this temperature than either single mutant. Among those growing well at 26.5°C, double mutants harboring drs2
and sec12-1 or sec23-1 also grew at 20°C, which is a nonpermissive temperature for drs2
single mutants. This suggests that sec12 and sec23 are able to partially suppress the cold-sensitive growth defect of drs2
, and that COPII may act upstream of Drs2p.
In stark contrast, chc1-ts drs2 double mutants were nearly always inviable. All of the spores that failed to grow in the tetrad analysis shown in Figure 1 B were predicted to be chc1-5 drs2
double mutants based on the genotype of the viable spores from each tetrad. The drs2
and chc1-5 single mutants grew nearly as well as wild-type progeny at this temperature (26.5°C, Figure 1 B; e.g., spores 3c, 3d, and 3b are drs2
, chc1-5, and wild-type, respectively). Microscopic examination of the plates indicated that the drs2
chc1-5 spores germinated and divided a few times to form microcolonies, indicating that mitotic growth, rather than germination, was impaired. In addition, this genetic interaction was not allele-specific since all three of the chc1 ts alleles tested were synthetically lethal with drs2
(Figure 1 A).
To further address the specificity of the synthetic lethal interaction between drs2, arf1
, and chc1-ts alleles, we crossed drs2
with several mutants that exhibit a defect in protein transport through the secretory or endocytic pathways. Double mutants combining drs2
with sec1-1, sec7-1, sec14-1, sec18-1, vps15
, vps21
, vps33
, vps35
, ypt7
, or end4-1 were obtained at the expected frequency, although the end4-1 drs2
, vps33
drs2
, and sec18-1 drs2
mutants exhibited a slow growth phenotype. Interestingly, a mutant allele of the yeast Eps15 homologue, pan1-20, was also synthetically lethal with drs2
(Figure 1 A). Eps15 is a cytoplasmic protein that localizes to clathrin-coated pits and may have an adaptor-like function in clathrin coated vesicle formation (
The drs2 Mutant Secretes Pro
-Factor at the Nonpermissive Temperature
One of the more striking phenotypes of yeast clathrin mutants is the mislocalization of several late Golgi (TGN) proteins that are required for pro-factor proteolytic processing. This results in the secretion of fully glycosylated pro
-factor rather than the mature peptide (
mutant, we would expect this mutant to secrete pro
-factor. To test this, wild-type, drs2
, and clc1
(clathrin light chain null) strains were grown at 30°C, and after shifting to 20°C for 1 h, were metabolically labeled and chased at this nonpermissive temperature for drs2
. Aliquots of cells were removed at the chase times indicated in Figure 2, converted to spheroplasts, and then centrifuged to separate intracellular (I) from extracellular (E) fractions.
-Factor was then recovered from each sample by immunoprecipitation.
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At the beginning of the chase, labeled -factor was present throughout the secretory pathway of the cells, as indicated by the presence of the core glycosylated ER proform, Golgi-modified hyperglycosylated proforms and the mature form (Figure 2, lanes 1, 7, and 13). In wild-type cells, complete proteolytic processing of pro
-factor occurred within 15 min, and most of the mature
-factor was secreted and degraded in the extracellular space. However, the clc1
cells were clearly deficient in the processing of pro
-factor, and most of the hyperglycosylated precursor was secreted into the extracellular space within 15 min, as previously reported (Figure 2, lane 18;
mutant secreted the hyperglycosylated pro
-factor and partially processed
-factor forms into the extracellular space (Figure 2, lanes 10 and 12). The drs2
mutant also exhibited a modest defect in Golgi-specific glycosylation since the hyperglycosylated pro
-factor secreted from drs2
cells showed a slightly faster mobility within the SDS-polyacrylamide gel relative to that from the clc1
strains. The kinetics of pro
-factor secretion at this temperature (20°C) was nearly equivalent to that of clc1
cells. These results indicate that late Golgi function is specifically perturbed in the drs2
cells and suggest a loss of clathrin function at the TGN at the nonpermissive temperature. In pulsechase experiments performed at 30°C, most of pro
-factor was processed and secreted as the mature form from drs2
cells (data not shown), indicating that this defect was temperature conditional. Moreover, the pro
-factor processing defect was observed within 15 min of preincubation at 20°C, suggesting a rapid onset of the temperature conditional phenotype (data not shown).
The drs2 Mutant Exhibits an Endosomal Defect
To examine protein transport through the endocytic pathway of drs2 cells, we followed the turnover of the
-factor receptor, Ste3p, which is constitutively endocytosed and delivered to the yeast vacuole where it is degraded (
, and arf1
cells were grown in galactose at 30°C to induce expression of the construct and populate the plasma membrane with the Ste3-myc protein. Glucose was added to the culture, which was then shifted to 15°C, and the disappearance of Ste3-myc was followed over time by immunoblotting. Even though the arf1
mutant displays an abnormal endosome morphology (
). In contrast, the rate of Ste3-myc turnover was three- to fivefold slower in the drs2
cells (Figure 3 A, drs2
) compared with wild type, suggesting a defect in endocytosis. To control for recovery of protein in each sample, the same blots were probed for carboxypeptidase Y (CPY), which was recovered equally in each sample (data not shown). drs2
cells cultured in glucose did not express Ste3p-myc, indicating that glucose repression of the GAL promoter was not perturbed (data not shown). Again, this phenotype was temperature conditional since the rate of Ste3-myc turnover at 30°C in the drs2
strain was only slightly (1.4-fold) slower than the wild-type strain (data not shown).
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Clathrin mutants display a similar defect in Ste3p turnover caused by inefficient internalization of the receptor from the plasma membrane ( cells by immunofluorescence localization of Ste3p-myc at each time point after glucose was added indicated an accumulation of Ste3-myc in intracellular structures with very little plasma membrane staining (data not shown). This result suggested that transport of Ste3p from the endosome to the vacuole, rather than internalization from the plasma membrane, was perturbed.
To more specifically test whether uptake from the plasma membrane or transport from endosomes to the vacuole was defective in drs2 cells at the nonpermissive temperature, we stained cells with the fluorescent endocytic marker FM4-64 (
cells (data not shown), and in wild-type cells it was delivered to the vacuole membrane in 3060 min (WT, 1 h). In contrast, the FM4-64 accumulated in endosomes of drs2
cells, which appear as punctate fluorescent spots in Figure 3 B (drs2
, 1 h). Even after 4 h at 15°C, most of the FM4-64 was not in the vacuoles and stained what appeared to be clusters of smaller structures (drs2
, 4 h). As the drs2
cells warmed up on the microscope stage, the FM4-64 was rapidly delivered to the vacuoles at all times tested, indicating that the defect was fully reversible (data not shown). In addition, vacuoles in drs2
cells that were stained with FM4-64 at 30°C did not appreciably fragment when the cells were shifted to 15°C (data not shown), so the structures stained at 15°C were indeed endosomes and not fragmented vacuoles.
These studies indicated that the endocytic defect observed in drs2 cells was attributable to a defect in the endosome-to-vacuole pathway rather than clathrin-dependent endocytosis from the plasma membrane. If so, CPY transport should be affected as well since this protein follows a TGN to endosome to vacuole delivery route. To test this, we examined the transport of CPY to the vacuole in the drs2
mutant. CPY is synthesized in the ER as the p1 precursor form and is modified on N-linked oligosaccharides by the Golgi
1,3 mannosyltransferase (Mnn1p) to form the p2 precursor. p2 CPY is sorted from secreted proteins in the TGN and ultimately processed to the mature form in the vacuole (
, and arf1
cells were pulse-labeled and chased at either the permissive or nonpermissive temperature of drs2
. Aliquots of cells were removed at the chase times indicated and CPY was recovered by immunoprecipitation. As shown in Figure 4, drs2
cells displayed near wild-type CPY transport kinetics at 30°C, while at 15°C the transport of CPY in drs2
mutants was significantly delayed relative to that in the wild-type cells, and was similar to the defect observed in arf1
cells at either temperature (approximately threefold slower transport kinetics). The partial glycosylation defect observed in drs2
(and arf1
cells) prevented the formation of a p2 CPY form that could be resolved from the p1 form in SDS-polyacrylamide gels. Thus, the kinetics of ER-to-Golgi transport could not be assessed for CPY. However, the ER-to-Golgi transport kinetics for
-factor and invertase in drs2
cells was found to be nearly wild type at the nonpermissive temperature, as scored by disappearance of the ER core form (Figure 2, and data not shown). Therefore, it is unlikely that ER-to-Golgi transport for CPY is disturbed in drs2
cells. These data are most consistent with the interpretation that protein transport from the late Golgi or endosomes to the vacuole is perturbed by the drs2
mutation. Interestingly, the chc1-5 allele isolated in the arf1
synthetic lethal screen also exhibits a partial glycosylation defect and an approximately threefold slower transport kinetics for CPY (
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The drs2 Mutant Accumulates Abnormal Membrane-bound Structures Similar to Berkeley Bodies
Most of the sec mutants that exhibit a temperature-conditional block in protein transport also accumulate an organelle or vesicular intermediate of the secretory pathway at the nonpermissive temperature. For example, the sec7 and sec14 mutations block protein transport out of the Golgi complex and accumulate Golgi structures called Berkeley bodies (
Morphological studies by electron microscopy revealed that drs2 cells accumulated aberrant double-membrane ring and crescent-shaped structures at both 15° and 30°C (Figure 5A and Figure B). The double-membrane rings in the drs2
cells measured 200250 nm in diameter (average, 240 nm) and often presented a significant gap between the concentric membranes, which should be equivalent to the luminal space of the cisternae. Representative double-membrane ring structures are marked with white arrowheads in Figure 5 A. Very similar ring structures also accumulated in the chc1
mutant (Figure 5 C;
cells (
cells were also modestly fenestrated (Figure 5 A, black arrow).
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To quantitate the effect of temperature on the accumulation of ring structures in wild-type and drs2 cells, these cells were grown at 26.5°C and shifted to the temperature indicated in Figure 5 E for 2 h before fixation. Regions containing 2325 cell sections were randomly selected and the number of crescent-shaped structures, double-membrane rings, and vesicles (50100-nm spheres) were counted and expressed as the number of structures per cell section. Relative to the wild-type strain, the drs2
mutant accumulated four- to eightfold more crescent and ring structures at the temperatures examined (Figure 5 E). In this analysis, structures similar to that shown in Figure 5 D were counted as rings in the wild-type cells. There was also a greater accumulation of these structures in the drs2
cells at the colder temperatures, which was particularly evident for the double-membrane rings. The number of vesicles in the drs2
cells dropped modestly at the colder temperatures, although within the range of values observed for the wild-type cells.
Golgi membranes that accumulate at the nonpermissive temperature in the sec7 mutant have been reported to become more extensively stacked in low glucose medium ( mutant grown in 2% glucose (Figure 5 B, arrow), but no significant difference was observed in the morphology of the membranes accumulating in drs2
cells incubated in low glucose (0.1%) medium for 2 h at the nonpermissive temperature. However, the effect of low glucose on Berkeley body structure in a sec7 mutant in our strain background was modest (data not shown).
Plasma membrane invaginations have been proposed to represent endocytic intermediates in yeast ( cells was observed under any of the growth conditions described above.
Reduction of Intact Clathrin-coated Vesicles that Can Be Isolated from the drs2 Mutant
A mammalian cytosolic ARF guanine nucleotide exchange factor requires both PS and phosphatidylinositol-4,5-bisphosphate for optimal membrane binding and subsequent activation of ARF ( cellular lysates by differential centrifugation to compare the amounts of ARF, clathrin, and adaptin subunits recovered in the 13,000-g pellets, and the 100,000-g pellets and supernatants. A small increase in the amount of clathrin heavy chain found in the 100,000-g supernatant fraction was occasionally observed in the mutant samples relative to the wild-type samples at both 15° and 30°C; otherwise, the fractionation pattern for clathrin was very similar between the two strains (data not shown). In addition, the fractionation pattern for ARF and the two adaptin small subunits (Aps1p for AP-1 and Aps2p for AP-2) was nearly identical between drs2
and wild-type samples. Therefore, the association of ARF and clathrin with bulk cellular membranes seemed to be unaffected in the drs2
mutant, although we cannot rule out the possibility that the distribution of ARF between specific organelles is perturbed.
To further assess clathrin function in the drs2 mutants, we asked whether CCVs could be purified from this mutant. CCV preparations were generated from drs2
and wild-type cells with or without a 1-h shift to 15°C (see Materials and Methods). Cell lysates were centrifuged at 21,000 g for 30 min to pellet large organellar membranes (e.g., plasma membrane, vacuolar membrane, ER, and mitochondria) and the resulting supernatant was centrifuged at 100,000 g to pellet vesicles. The 100,000-g pellet was then applied to a Sephacryl S-1000 gel filtration column (
samples from cells shifted to 15°C (Figure 6 A) or 30°C (Figure 7 D, and data not shown). In addition, from Coomassie bluestained gels, we estimated that the recovery of clathrin heavy chain in these fractions was comparable for both strains at both temperatures (data not shown).
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However, a clear difference between wild-type and mutant samples was evident when fractions highly enriched for the clathrin heavy chain were examined by EM. Peak clathrin fractions obtained from wild-type cells were substantially enriched in CCVs with a clear lipid bilayer present in a substantial number of the vesicle profiles (Figure 6 C, arrowheads). In contrast, most of the vesicle profiles in the drs2 peak clathrin fractions contained an electron dense center, but no lipid bilayer within the clathrin basket (Figure 6 D, open arrowheads). In addition, the drs2
fractions contained what appeared to be partially assembled clathrin lattices (Figure 6 D, arrow) that were rarely observed in wild-type fractions. CCVs with a lipid bilayer were occasionally observed in the drs2
fractions, but were found at <1/10 of the frequency per section as compared with the wild-type samples (multiple sections from three different preparations were compared). This was a temperature-conditional phenomenon as CCV preparations from drs2
cells maintained at a permissive temperature were indistinguishable from wild-type samples (data not shown).
It is well established that clathrin triskelions can self assemble into baskets in slightly acidic, low ionic strength buffers ( cell lysate. It appears that these clathrin structures pellet at 100,000 g and elute from the S-1000 column in similar fractions as bona fide CCVs. The simplest interpretation of these data is that the drs2
cells are deficient in producing CCVs at the nonpermissive temperature. It is also possible that clathrin dissociates more readily from CCVs produced in drs2
cells and reassembles into baskets during purification, thus preventing the isolation of bona fide CCVs. In either case, there is a marked difference between drs2
and wild-type cells either in the ability to produce CCVs or in the physical properties of CCVs from these strains.
Even though most large membranes pellet at 21,000 g, we had noticed by EM that the 100,000-g pellet from drs2 cells (that was applied to the S1000 column) contained a significant number of aberrant membrane structures that were similar in appearance and size to those observed in cell sections (Figure 5 B). Because drs2
cells exhibited both Golgi and endosome-associated defects, it was possible that both organelles would accumulate in the drs2
mutant. Therefore, we examined the S1000 column fractions from the CCV preparations for membranes containing a late Golgi protein,
1,3-mannosyltransferase (Mnn1p), and an endosomal t-SNARE, Pep12p (Figure 6 A). In fractions from wild-type cells, Mnn1p eluted slightly later than clathrin, which likely represents small vesicles or small Golgi fragments. However, a substantial change in the elution profile of Mnn1p-containing membranes was observed in fractions from drs2
cells. A peak of Mnn1p eluted just after the void volume and spread from fractions 1631. This change in Mnn1p distribution was observed with samples prepared from drs2
cells incubated at both 15°C (Figure 6 A) and 30°C (data not shown), and correlated with the large membrane structures observed by EM in these fractions (e.g., Figure 6 B, fraction 16). Membranes of any sort were difficult to find by EM in fraction 16 from wild-type samples (data not shown). Relative to Mnn1p, a wider distribution of Pep12p-containing membranes was found in the S1000 fractions from wild-type cells. However, the Pep12p elution profile was not significantly different between the wild-type and drs2
fractions (Figure 6 A). These data suggest that the abnormal membrane structures that accumulate in the drs2
cells are Golgi membranes and not endosomal membranes.
Drs2p Localizes to Late Golgi Membranes Containing Kex2p and Mnn1p
To analyze the intracellular localization of Drs2p, we prepared a rabbit polyclonal antiserum against a bacterially expressed fragment of Drs2p (amino acid residues 528920). Affinity-purified antiDrs2p antibodies recognized an ~150-kD protein on an immunoblot of a wild-type cell lysate (Figure 7 A, DRS2), which is in close agreement to the predicted mass of 154 kD. This protein was not present in a drs2 cell lysate and was found in greater abundance in wild-type strain carrying DRS2 on a multicopy plasmid (Figure 7 A, drs2
and 2 µ DRS2). Thus, the 150-kD protein is equivalent to Drs2p.
Drs2p was recently suggested to localize to the plasma membrane based on cofractionation of an epitope-tagged Drs2p with the plasma membrane ATPase, Pma1p, in sucrose gradient fractions from a crude cell lysate (
The fractionation profile for Drs2p shown in Figure 7 B is very similar to how CCV and late Golgi proteins such as Kex2p fractionate. To further characterize the localization of Drs2p, a P100 fraction was applied to the bottom of a sucrose gradient and centrifuged to equilibrium (
To determine whether Drs2p and Kex2p reside in the same membrane structures, wild-type cells overexpressing a hemagglutinin (HA)-tagged Kex2p were stained simultaneously with antibodies to Drs2p and HA. Localization of Drs2p by immunofluorescence produced a punctate staining pattern that is typical for the yeast Golgi complex (Figure 8, WT + 2µ KEX2HA, -Drs2p). Although the signal was weak, it was clearly above the background staining observed for the drs2
strain (Figure 8, drs2
). The HA antibody also produced a punctate staining pattern (Figure 8, WT + 2µ KEX2HA,
-HA) and, importantly, most of the structures that were positive for Drs2p also stained for Kex2-HA. As expected, very little overlap was observed between Drs2p and the early Golgi marker Och1-HA (data not shown).
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We could not detect plasma membrane staining with the Drs2p antibodies, which is inconsistent with a previous report suggesting that Drs2p resides in the plasma membrane (1,3-linked mannose residues that are found on many glycoproteins. For these cells, staining of the plasma membrane is clearly observed as a stained rim around the cells (Figure 8, anti
1,3). Thus, by both immunofluorescence and subcellular fractionation studies, it appears that most, if not all, of Drs2p is found in late Golgi membranes.
Mutation of Aspartic Acid 560 Abolishes Drs2p Function In Vivo
Drs2p is predicted to be a P-type ATPase based on the presence of five well-conserved ATPase motifs involved in the binding and hydrolysis of ATP (Figure 9 A). For P-type ATPases, an aspartic acid within the second motif forms an aspartyl-phosphate catalytic intermediate that is essential for ATP hydrolysis ( strain. These mutations would be expected to cause minimal structural changes in Drs2p, but should abolish the presumed ATPase activity of this protein. The drs2
strain carrying either the wild-type DRS2 gene (Wild-type), an empty vector (drs2
), or two independent isolates of each mutant (D560N and D560E) were tested for growth at 30° and 20°C (Figure 9 B). As previously reported, the drs2
strain failed to grow at 20°C, but grew well at 30°C. None of the strains carrying the D560 point mutations were able to grow at 20°C (Figure 9 B), even though each of the strains expressed a wild-type level of Drs2p (data not shown). These data support the assignment of Drs2p as a P-type ATPase and suggest that the ATPase activity of Drs2p is essential for its function in vivo.
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Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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In this report, we present several lines of evidence that strongly implicate the integral membrane P-type ATPase Drs2p in late Golgi function, and suggest a link between Drs2p and CCV formation from Golgi membranes. (a) A specific synthetic lethal interaction was found between drs2, arf1
, and chc1-ts alleles. (b) The drs2
mutant exhibits a cold-sensitive defect in the proteolytic processing of pro
-factor in the yeast TGN that is comparable with the defect shown by clathrin mutants. (c) The drs2
mutant accumulates aberrant Golgi structures that are morphologically comparable to the membrane structures that accumulate in clathrin mutants. (d) We observed a substantial decrease in the yield of CCVs that could be isolated from the drs2
mutant preincubated at the nonpermissive temperature. (e) Drs2p localizes to late Golgi membranes containing Kex2p.
Specific Genetic Interactions between drs2, arf1
, and chc1-ts Alleles
Our rationale for performing the arf1 synthetic lethal screen was that it could provide an unbiased, in vivo approach to identify proteins that regulate ARF function, or participate with ARF in CCV or COPI vesicle formation. This was the first yeast genetic screen we are aware of that has uncovered a mutant allele of the clathrin heavy chain gene (swa5-1/chc1-5;
strain (
. This provides genetic support for the substantial biochemical evidence in the literature implicating ARF in clathrin recruitment to Golgi membranes (
synthetic lethal approach.
We were initially concerned about the specificity of the drs2arf1
synthetic lethal interaction, even though four alleles of the SWA3/DRS2 gene were recovered in this genetic screen. However, from crosses with 19 different mutants that perturb the secretory or endocytic pathways, we found a specific interaction between pairwise combinations of arf1
, drs2
, and chc1-ts alleles. The drs2
allele was also synthetically lethal with pan1-20, an eps15-related protein that interacts with yAP180 (yeast homologue of clathrin assembly protein AP180) in a yeast two-hybrid assay (
and mutant ts alleles encoding three different COPI subunits, even though combination of arf1
with these COPI alleles produces a synthetic growth defect (
Phenotypes Exhibited by the drs2 Mutant
These genetic analyses prompted us to examine the drs2 mutant for defects in the secretory and endocytic pathways. The secretion kinetics and Golgi-specific glycosylation of pro
-factor and invertase were only modestly perturbed. However, a substantial cold-sensitive defect in the Kex2p-dependent processing of pro
-factor was observed. This resulted in secretion of the pro
-factor precursor, a phenotype also exhibited by clathrin mutants (
mutation seems to specifically perturb late Golgi (TGN) and endosome function.
As visualized by EM, the drs2 mutant accumulates abnormal membrane-bound structures that were morphologically equivalent to structures that accumulate in clathrin mutants. The abnormal membrane structures that were isolated from drs2
cells by differential centrifugation and gel exclusion chromatography were enriched for a late Golgi protein, but not for an endosomal marker protein. Since the localization studies suggest that Drs2p is a late Golgi (TGN) resident, these data strongly suggest that Drs2p acts in the late Golgi to maintain normal structure and function of this compartment. It is possible that Drs2p has some function in the endosome as well, or the effect on endosome function could be a secondary consequence of perturbing late Golgi function.
Perhaps the most compelling evidence that Drs2p plays a role in clathrin function is the striking difference in the appearance of CCV preparations between drs2 and wild-type cells. Very few bona fide CCVs could be isolated from drs2
cells preincubated at the nonpermissive temperature. These preparations contained clathrin baskets and lattices with no associated membrane. In contrast, CCV preparations from drs2
cells maintained at 30°C were indistinguishable from wild-type samples. These results suggest that Drs2p is required to form clathrin-coated vesicles from Golgi membranes at temperatures below 23°C. However, it is also possible that the association of clathrin coats with vesicle membranes from drs2
cells is less stable and the coat dissociates during cell lysis. In either case, loss of Drs2p clearly perturbs CCVs.
At this time, it is not possible to distinguish whether the effect of Drs2p on clathrin function is direct or a secondary consequence of the abnormal TGN structure. However, this effect is specific since the TGN of drs2 cells functions normally in the ability to sort vacuolar proteins and in protein secretion, despite the abnormal morphology. This suggests that late secretory vesicles bud normally from the TGN in drs2
cells. The genetic interactions also showed a high degree of specificity between drs2
and clathrin mutations. Therefore, there is not a wholesale loss of Golgi function in drs2
cells at the nonpermissive temperature. Indeed, most of the specific defects observed can be explained by a loss of clathrin function. Even the accumulation of abnormal membrane at the permissive temperature could be the result of inefficient CCV budding.
The temperature-conditional defects observed for a strain carrying a complete loss of function allele is somewhat unusual. This suggests that Drs2p is required to overcome an inherently cold-sensitive process in the cell (perhaps CCV budding), such that the growth defect is caused by the combination of low temperature and loss of Drs2p. However, this cold-sensitive process is not at the extreme of the normal growth range for yeast; drs2 cells fail to grow at room temperature (20°C) and the mutant phenotypes described here are observed at this temperature. In addition, some of the drs2
phenotypes, such as arf1
synthetic lethality and abnormal Golgi morphology, are also observed at 30°C. Thus, it appears that Drs2p plays a role in Golgi function at all temperatures examined but is only essential below 23°C. Alternatively, it is possible that Drs2p function is essential at all temperatures, but loss of Drs2p is compensated by one or more of the Drs2p-related P-type ATPases at higher temperatures. In either case, Drs2p clearly plays a critical role for organisms such as yeast since the ability to adapt to daily fluctuations in temperature is essential for their survival.
Potential Function of Drs2p as an Aminophospholipid Translocase
What is the biochemical function of Drs2p? Many P-type ATPases use the energy of ATP hydrolysis to pump cations such as Ca2+, H+, Na+, or heavy metals across a membrane against their electrochemical gradient. These transporting ATPases contain signature motifs that allowed the identification of 16 P-type ATPases in the yeast genome that can be phylogenetically grouped into six distinct families (
The drs2 mutant has been reported to exhibit a defect in the translocation of a fluorescent PS derivative across the plasma membrane (
cells in the translocation of fluorescent lipid derivatives across the plasma membrane. This group also found no difference in the amount of PE exposed on the outer leaflet of the plasma membrane as detected by trinitrobenzene sulfonic acid labeling. These latter observations could suggest that Drs2p is not an aminophospholipid translocase, but it is more likely that the Drs2p protein is simply not present at the plasma membrane and therefore is not required to maintain an asymmetric distribution of aminophospholipids in this membrane. In fact, we cannot detect Drs2p at the plasma membrane by immunofluorescence localization or subcellular fractionation (Figure 7 and Figure 8). Measurement of translocase activity with Golgi membrane fractions is more complicated because the direction of flip is expected to be from the luminal to the cytoplasmic leaflet and thus requires incorporation of the probe into the luminal leaflet. Thus, further work is required to determine if Drs2p is an aminophospholipid translocase, as suggested by its homology to ATPase II.
Since an aminophospholipid translocase activity is the only biochemical function attributed to ATPase II and Drs2p in the literature, it is relevant to speculate on how membrane asymmetry may affect Golgi structure and perhaps clathrin function. The bilayer couple hypothesis of mutant, which are notable for their lack of fenestration or tubular regions. Particularly in comparison to the arf1
mutant in which the Golgi is highly fenestrated or tubular in appearance (
Others have proposed that the transbilayer movement of lipid could induce the bending of membranes to facilitate vesicle budding (
A third possibility is that an increase in the PE or PS concentration of the cytoplasmic leaflet may influence the recruitment or activity of peripherally associated proteins. For example, the association of an ARF guanine nucleotide exchange factor with Golgi membranes might be influenced by the PS concentration (
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Footnotes |
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Drs. Chen and Ingram contributed equally to this work and should be considered cofirst authors.
1 Abbreviations used in this paper: AP, adaptor protein; ARF, ADP-ribosylation factor; CCV, clathrin-coated vesicle; CPY, carboxypeptidase Y; EM, electron microscopy; HA, hemagglutinin; ORF, open reading frame; PE, phosphatidylethanolamine; PS, phosphatidylserine; ts allele, temperature-sensitive allele.
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Acknowledgements |
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We thank Scott Emr, Richard Kahn, Sandra Lemmon, Greg Payne, Erin Gaynor, Bruce Horazdovsky, Beverly Wendland, and Todd Brigance for antibodies, strains, or plasmids. We thank Todd Reynolds for providing sucrose gradient fractions (for Figure 7 C). We are particularly grateful to Guang-Ming Wu, Sidney Fleischer, and Paula Flicker for assistance with electron microscopy. We also thank Greg Payne, Sandra Lemmon, and members of the Graham lab for their helpful comments during the course of these experiments.
This work was supported by grants from the National Science Foundation (MCB-9600835, BIR-9419667) to T.R. Graham.
Submitted: 11 May 1999
Revised: 5 November 1999
Accepted: 9 November 1999
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