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An arf1
Synthetic Lethal Screen Identifies a New Clathrin Heavy Chain Conditional Allele That Perturbs Vacuolar Protein Transport in Saccharomyces cerevisiae
Chih-Ying Chena and
Todd R. Grahama
a Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235
Corresponding author: Todd R. Graham, Department of Molecular Biology, Box 1820 Station B, Vanderbilt University, Nashville, TN 37235., grahamtr{at}ctrvax.vanderbilt.edu (E-mail).
Communicating editor: E. W. JONES
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1 allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1). Most of the swa mutants exhibit phenotypes comparable to arf1 mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.
PROTEIN transport between distinct organelles in eukaryotic cells is carried out by membrane-bound vesicles. Formation of transport vesicles involves assembly of cytosolic coat proteins onto the donor membrane, selective packaging of the cargo proteins and finally budding of the coated vesicles. Several types of vesicle coats that mediate different protein transport steps have been studied in detail (reviewed in 
,ß,ß',
,
,
,
-COP) protein complex, coats COPI vesicles that mediate transport from the Golgi to the ER and between Golgi compartments. Two types of coat proteins contain clathrin, which is composed of heavy chains and light chains, associated with different adaptor protein (AP) complexes. In mammalian cells, clathrin/AP-1 coated vesicles bud from the trans-Golgi network and deliver cargo to endosomal compartments, while clathrin/AP-2 drives endocytosis from the plasma membrane. Recently, a third adaptor complex (AP-3) has been identified (
Small GTP-binding proteins are required to initiate (or prime) assembly of the coats. As Sar1p functions to recruit COPII to the ER membrane, ADP-ribosylation factor (ARF) appears to be required on the Golgi membrane to recruit COPI or AP-1/clathrin coats (S-activated form of ARF (S-activated ARF can also mediate recruitment of AP-2 and COPI to endosomal membranes (
In yeast Saccharomyces cerevisiae, ARF is encoded by two genes, ARF1 and ARF2, which encode proteins with 96% identity that are probably redundant in function ( arf2 mutants are inviable, indicating that ARF is an essential protein in yeast. The Arf2 protein is only expressed at 10% of the level of Arf1 protein, and strains carrying a deletion of the ARF2 gene show a wild-type phenotype ( mutants exhibit a substantial alteration in the structure of Golgi and endosomal compartments ( null mutation combined with mutations in RET1, SEC21, and SEC27, which respectively encode -, -, and ß'-COP subunits of the coatomer complex, were found to display a synthetic growth defect, whereas double mutants harboring arf1 combined with several other sec mutations do not exhibit synthetic growth defects (
To identify other proteins that functionally interact with Arf1p, we have employed a genetic screen to search for the mutations that display synthetic lethality in combination with the arf1 null mutation and have identified seven complementation groups (SWA1-7 for synthetically lethal with arf1). In this study, we focused on characterization of SWA5, which was found to be allelic to the clathrin heavy chain gene (CHC1). This genetic interaction between arf1 and chc1 provides in vivo support for a role for ARF in clathrin coat assembly. Surprisingly, chc1-5 (swa5-1) mutants exhibited markedly slow transport kinetics for carboxypeptidase Y (CPY) that was not observed in three other chc1 temperature-sensitive (ts) mutants. The effect of chc1-5 on CPY transport was much more substantial than the effect on invertase or alkaline phosphatase transport, further implicating clathrin in transport of CPY from the Golgi to the vacuole.
| MATERIALS AND METHODS |
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Media, strains, and plasmid construction:
Yeast strains and plasmids used in this study are listed in Table 1. CCY2011C and CCY2804C are meiotic progeny from crosses of CH1305 ( and of CH1304 ( (
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Plasmids pCC8218 and pCC616 were generated as follows: a 1.8-kb EcoRI-PstI fragment carrying ARF1 was subcloned from pRB1297 (
The isogenic chc1-ts strains were prepared by targeted integration of 5' truncated chc1 alleles into the CHC1 locus (depicted in Figure 7B). YIpchc521Cla (57 was subcloned from p57 (57. The 5' ends of the chc1 genes were deleted by digesting the pCC306 plasmids with BglII and ClaI, blunt-ending and ligating to generate the 3' chc1 series of plasmids. To prepare p 3'chc1-43, two PCR primers were designed to delete the C-terminal 43 amino acids of Chc1p: one is upstream of the last BamHI site in the CHC1 open reading frame (5'-ACAAATTTGACCAATTGGGATTG-3'); the other introduces two stop codons and a SalI site after codon 1610 (5'-CGTCGAGTCGACTCATTATTTTTTTATGGAGATTTCAAATGGC-3'). After PCR amplification, the products were digested with SalI and BamHI and subcloned into the SalI-BamHI sites of p 3'chc1-5. To generate the 6210 chc1 mutants, the p 3'chc1 plasmids were linearized with AftII and transformed into SEY6210 to target integration into the chromosomal CHC1 locus. Ura+ transformants were tested for ts growth. Total DNA was then isolated from ts transformants and the correct integration event was confirmed by PCR and DNA sequencing.
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Isolation and characterization of swa mutants:
To start the screen, CCY2011 and CCY2804 were grown at 30° in liquid SD medium lacking uracil to stationary phase and treated with 3% ethyl methanesulfonate (Fluka, St. Louis) for 30 min at 30° (
To test for complementation or suppression of swa mutants by known genes, pEP1 (2µ GCS1 LEU2 AmpR, a gift from G. C. Johnston, Dalhousie University, Canada), pCLJ80 (2µ GEA1 LEU2 AmpR;
To clone SWA5, a genomic library (
Cell labeling, immunoblotting, and invertase assay:
Pulse-chase metabolic labeling of cells with 35S amino acids and immunoprecipitations was done as previously described (
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolation of mutants that require Arf1p for survival:
To better understand the primary function of ARF in vivo, we performed a genetic screen to search for the genes that, when mutated in an arf1 null background, lead to a lethal phenotype. An ade2 ade3 based colony-sectoring assay ( ade2 ade3 mutations (CCY2804C and CCY2011C) are white, while the transformants carrying wild-type ARF1 and ADE3 genes linked together on a plasmid (pCC8218, URA3-based) form red colonies that contain white sectors (Figure 1A). This sectoring phenotype indicates the cells can readily lose the ARF1-ADE3-URA3 plasmid, which is also demonstrated by the ability of these strains to grow on medium containing FOA, a drug that selects against cells harboring the URA3 gene. Among ~19,000 colonies that survived ethyl methanesulfonate treatment, 63 mutant colonies consistently showed a nonsectoring red phenotype on YPD medium (Figure 1A) and no growth on FOA at 30°, indicating that these strains could not survive without the plasmid. Three mutants were discarded because they were unable to lose the original ARF1-ADE3-URA3 plasmid when transformed with ARF1 carried on a LEU2-based vector (pCC616), suggesting the mutations that caused the nonsectoring phenotype were independent of Arf1p.
Because protein secretion is an essential process, mutations in genes involved in the secretory pathway usually affect the growth of the mutants. We screened for mutants that grew well at 30°. However, it was possible that these mutants would exhibit a conditional growth defect. Because the nonsectoring mutants harbor a copy of wild-type ARF1 on a plasmid, additional phenotypes, such as temperature conditional growth, should be attributable to the swa mutations. Therefore, all 60 of the mutants were examined for cold sensitive (cs) or temperature sensitive (ts) defects in growth at 15° or 37°, respectively. Thirty-five of the mutants were either cs, ts, or both.
From backcrosses to parental strains it was determined that all the mutants contained recessive mutations (see MATERIALS AND METHODS). Four complementation groups each containing two or more alleles were defined (SWA1-SWA4, Figure 2A) by intercrossing the mutants and testing for complementation of the nonsectoring phenotype in diploids. From this analysis, 47 mutants remained unassigned, including 27 that exhibited a conditional growth defect.
All of the strains displaying a conditional growth phenotype were analyzed directly for protein transport defects by pulse-chase experiments at their nonpermissive temperature and immunoprecipitation of the vacuolar protein CPY (described in detail below). Ten of the 27 unassigned conditional mutants displayed a defect in CPY transport or glycosylation and were characterized further to determine if this phenotype was caused by a single gene mutation. The mutants were backcrossed with the parental strain, diploids were sporulated, and tetrads were dissected. During the tetrad analyses, we found that the ARF1-ADE3-URA3 plasmid was frequently lost during meiosis and not recovered in the progeny. For example, instead of finding two sectoring vs. two nonsectoring progeny from a heterozygous diploid carrying a single gene mutation, two viable (white, arf1 SWA) vs. two dead (arf1 swa) progeny were usually observed (Figure 1B). Three more complementation groups were confirmed by the tetrad analyses, each containing one mutant allele, and designated as SWA5-SWA7. Seven of the temperature conditional mutants did not show 2:2 segregation of the synthetic lethal phenotype, indicative of multiple mutations, and thus were not characterized further (see MATERIALS AND METHODS). For swa5-1, ~50% of the predicted double mutant progeny were not able to survive without the plasmid carrying ARF1 (Figure 1B). The surviving swa5-1 arf1 double mutants grew extremely poorly relative to swa5-1 or arf1 single mutants, which indicated that the genetic interaction between swa5-1 and arf1 ranged from a strong synthetic growth defect to lethality.
The pulse-chase analyses were repeated to examine the transport kinetics of CPY for representative mutants from each complementation group that exhibited a conditional growth defect. The vacuolar hydrolase CPY is initially synthesized in the ER as a core-glycosylated p1 proenzyme, which is then converted to a p2 form in the Golgi complex by modification of core oligosaccharides and subsequently processed to the mature form (mCPY) upon arrival in the vacuole ( cells. The swa3-2, swa4-2, swa6-1, and swa7-1 mutants exhibited a defect in CPY transport comparable to that observed in arf1 cells (approximately threefold delay in transport, marked as ++). swa6-1 consistently incorporated significantly less [35S]methionine into TCA precipitable proteins than the other mutants and is not shown in Figure 2. The swa1-2 mutant was indistinguishable from wild type (++++), whereas swa2-2 mutants displayed a modest but reproducible transport defect (+++). The swa5-1 mutant displayed the strongest defect in CPY transport, with a three- to sixfold increase in the half time of CPY maturation (+). For swa3-2, swa4-2, and swa5-1, a slight CPY transport defect was apparent at 30° but was exaggerated at the nonpermissive temperature.
As described above, several of the swa mutants displayed cs growth and protein transport defects that are very similar to those observed in arf1 cells ( mutants ( mutants can grow on 40 mM but not 60 mM NaF. Interestingly, swa3 mutants (swa3-1 and swa3-2) were extremely sensitive to fluoride and were not able to grow on the lowest concentration tested (20 mM). swa4-1, swa5-1, and swa7-1 mutants were as sensitive to fluoride as the arf1 mutants, whereas swa2-2 exhibited a sensitivity intermediate to swa3-2 and arf1 (Figure 2A). Again, the swa1-2 mutants behaved more like the wild-type strain (Figure 2A).
Mutations in SEC7, SEC21 (-COP), GEA1 (ARF-guanine nucleotide exchange factor), and GCS1 (ARF-GTPase activating protein) were previously found to display genetic interactions with the arf1 mutation (
The clathrin heavy chain (CHC1) gene complements the ts and protein transport defect of swa5-1 mutants:
Since the swa5-1 mutant exhibited a ts growth defect and markedly slow CPY transport kinetics, we decided to first focus on SWA5 characterization. The SWA5 gene was cloned by complementing the ts phenotype of the mutant. From a yeast low-copy genomic library, we isolated a plasmid carrying an ~10.8-kb insert from chromosome VII (pCC1-2) containing two open reading frames, CDC68 and CHC1 (Figure 3A). Deletion of a 1735-bp fragment in CHC1 (NsiI) disrupted the ability of this plasmid to complement the growth defects of swa5-1 mutants at 37° while a fragment containing only full-length CHC1 complemented the ts phenotype (Figure 3, A and B). Even though ARF has been implicated in AP-1/clathrin recruitment to Golgi membranes, this was a surprising result because CPY transport has been characterized in chc1 mutants previously with no defect observed in a null mutant (
The swa5-1 mutation resides within CHC1:
To determine whether SWA5 is allelic to CHC1, gap rescue (AatII-BamHI) did not complement the swa5-1 ts growth defect, indicating that a mutation(s) was located within this region. DNA sequencing revealed that a G at nucleotide position 4831 is missing, which results in a frameshift mutation 43 codons before the end of the open reading frame and consequently a truncated clathrin heavy chain (Chc1p) with 28 missense amino acids at the C terminus (see Figure 7A). Since these data indicated that the swa5-1 mutation resides in the CHC1 gene, we will now refer to the swa5-1 allele as chc1-5.
Immunoblotting was performed to examine the stability of the mutant Chc1p. Strains harboring chc1-5, chc1-521, or chc1- alleles and wild-type strains were grown to mid-log phase at 23° and then shifted to 37°. Lysates from cells taken at the indicated time points were subjected to SDS-PAGE, blotted, and then probed with Chc1p and CPY antibodies, the latter to control for equal loading of the gel. As expected from the chc1-5 mapping and sequencing data, the mobility of the mutant protein was indistinguishable from that of the wild-type Chc1p. However, the level of Chc1-5 protein was reduced in cells growing at a permissive temperature and dropped substantially when the cells were shifted to 37° (Figure 5, lanes 4–6). Interestingly, the level of wild-type Chc1p also dropped after cells were shifted to 37° for 1 hr and increased somewhat by 2 hr (Figure 5, lanes 1–3). The loss of mutant Chc1p after temperature shift appeared more gradual for the chc1-521 mutant even compared to the isogenic wild-type strain (Figure 5, lanes 7–12).
The genetic interaction between arf1 and chc1 is not allele specific:
To test for a synthetic interaction of chc1-5 with arf1 mutations other than arf1, two arf1 mutant alleles carried on plasmids ( mutants at 30° but not at 37°, suggesting that the encoded Arf1p is partially defective. Progeny of the cross between arf1 pJCY1-58 (or pJCY1-62) and chc1-5 mutants were characterized by random spore analyses. The [W66R] arf1 allele can efficiently rescue the synthetic lethality between chc1-5 and arf1, because an equal number of arf1 chc1-5 and arf1 SWA5 segregants were recovered among the progeny that retained the plasmid. However, a modest growth defect was observed for the [W66R] arf1 chc1-5 double mutants at 30° that was not observed for the parental single mutant strains (data not shown). In contrast, no chc1-5 arf1 double mutants carrying [D26G] arf1 were recovered among 67 progeny tested, indicating that [D26G] arf1 cannot rescue the synthetic lethal interaction between arf1 and chc1-5. Therefore, the genetic interaction between arf1 and chc1-5 is not restricted to the arf1 allele, but is also observed for at least one point mutation in ARF1. In addition, crosses between strains harboring arf1 and other chc1 alleles (chc1-521 and chc1-57) have been performed to construct double mutants. Although the frequency of viable double mutant progeny from these crosses appeared greater than for arf1 chc1-5, the viable arf1 chc1-521 and arf1 chc1-57 strains also grew extremely poorly (data not shown).
The chc1-5 mutant mislocalizes Golgi enzymes to the plasma membrane:
Previous studies showed that clathrin function is required at late Golgi compartments for retaining resident Golgi enzymes, like Mnn1p, Kex2p, and dipeptidylaminopeptidase A, which are mislocalized to the plasma membrane of chc1 mutants (-factor and secretion of this precursor form from chc1 mutants. We also found that the chc1-5 mutant (CCY620-5) secreted -factor precursor at the nonpermissive temperature to the same extent as other chc1 mutants (data not shown). To directly determine if Golgi enzymes were mislocalized to the plasma membrane of the chc1-5 mutant incubated at the nonpermissive temperature, perhaps causing the glycosylation defects (Figure 3C), we analyzed the extent of mislocalization of the fusion protein M39I, containing the reporter enzyme invertase attached to the cytoplasmic tail and transmembrane domain (Golgi-localization signal) of Mnn1p. Wild-type and chc1-5 cells expressed 5–7% of M39I on the plasma membrane at the permissive temperature (Figure 6, 0 hr). In wild-type cells, the percentage of M39I in the plasma membrane did not change during a 2 hr incubation at 37°. In contrast, chc1-5 cells mislocalized ~2.5-fold more M39I to the plasma membrane after temperature shift (Figure 6). These phenotypes exhibited by the chc1-5 mutant, secretion of -factor precursor and mislocalization of a Golgi-retained reporter protein to the plasma membrane, are similar to those previously described for other chc1 mutants.
The CPY transport defect exhibited by the chc1-5 mutant is allele-specific:
The chc1-5 mutant exhibited a kinetic defect in CPY transport that has not been observed in strains harboring other mutant alleles of chc1 (57; chc1*) were constructed, linearized, and transformed into SEY6210 to simultaneously disrupt the wild-type CHC1 gene and integrate the mutant alleles (Figure 7B). All the resulting chc1 mutants were ts for growth. The isogenic strains were grown at 23°, shifted to 37° for 1 hr, and subjected to pulse-chase labeling to analyze CPY transport. Mutants harboring chc1-521 and chc1-57 exhibited CPY transport kinetics similar to those of the wild-type strain, but the chc1-5 mutants still displayed the slow transport defect (Figure 7C). These results indicate that the chc1-5 mutant allele indeed causes the slow CPY transport and other chc1 ts alleles do not affect CPY transport in the SEY6210 background after 1 hr preincubation at 37°.
Although chc1-5 and chc1-57 encode similar mutant forms of the clathrin heavy chain, the effects of these mutations on CPY transport were quite different. To determine if this phenotypic difference was caused by the addition of 28 missense amino acids or by the loss of the C-terminal 43 amino acids, we introduced a stop codon at position 1611 to produce the chc1-43 allele (Figure 7A). The chc1-43 mutation also causes a ts growth defect in the SEY6210 background, similar to the other isogenic chc1 mutants generated. As shown in Figure 7C, chc1-43 mutant cells exhibited normal transport kinetics for CPY but did exhibit a partial defect in glycosylation, as p2 CPY was not resolved well from p1 CPY. Therefore, it appears that it is the 28 missense amino acids at the very C terminus of the Chc1-5 protein that interfere with transport, rather than the lack of the C-terminal 43 amino acids. Furthermore, the inefficient conversion of p1 to p2 CPY in the chc1-5 mutant gives the impression that transport from the ER to Golgi is the slow transport step. However, the chc1-43 mutant exhibits a similar glycosylation defect but normal transport kinetics for CPY, suggesting that the apparent accumulation of p1 CPY in chc1-5 cells is caused by underglycosylation rather than a defect in ER-to-Golgi transport. This glycosylation defect is probably due to the mislocalization of 1,3 mannosyltransferase (Mnn1p), which is primarily responsible for the conversion of p1 to p2 CPY (
chc1-5 specifically perturbs the vacuolar protein transport route taken by CPY:
There are at least three known routes for transfer of newly synthesized proteins out of the Golgi complex: (1) secretion to the cell surface; (2) transport via endocytic compartments (or endosomes) to the vacuole, which is the route taken by CPY and is perturbed in vps mutants; and (3) the route taken by alkaline phosphatase (ALP) to the vacuole, which is not perturbed in most vps mutants (
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| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Identification of mutations that exhibit synthetic lethality with arf1:
We have initiated a genetic screen to find factors that functionally interact with ARF and have defined seven complementation groups of swa mutants. Strains harboring the arf1 null allele exhibit phenotypes of cs growth, hypersensitivity to fluoride ions, an approximately threefold delay in the kinetics of protein transport through the secretory pathway, and Golgi-specific glycosylation defects ( mutant. The basis of the screen, synthetic lethality between swa and arf1 mutations, also strongly implicates the SWA genes in ARF function in vivo. Of particular note, the swa3 cs mutants grow poorly at temperatures as high as 23° (data not shown) and are also extremely hypersensitive to fluoride ions (Figure 2A). The cold and fluoride sensitivity of the arf1 mutant could be explained by partial loss of SWA3 function if ARF is an upstream activator of Swa3p.
Genes previously found to display genetic interactions with ARF1, such as SEC21, SEC7, GEA1, and GCS1, were apparently not represented among the seven complementation groups defined here. This could be due to differences in the extent of the synthetic defect exhibited by distinct genetic strains. For instance, coatomer mutants were not found to be synthetically lethal with arf1 in the SEY6210 background ( arf2 mutants are inviable, ARF2 is a gene that should be identified in the screen. However, diploid strains of the genotype ade2/ade2 ade3/ade3 arf1/arf1 ARF2/arf2 pADE3 ARF1 were not able to sector (data not shown), suggesting that a single copy of wild-type ARF2 is insufficient for a diploid cell to live. Therefore, strains carrying mutations in the ARF2 gene in this screen would appear as harboring dominant mutations. Since all of the swa mutations tested were recessive, it is unlikely that ARF2 is any of the SWA genes. This is difficult to test directly because transformation of the swa mutants with ARF2 carried on a CEN plasmid suppresses the nonsectoring phenotype of most of the mutants. The fact that a single copy of ARF2 rescues the synthetic lethality between arf1 and the swa mutations indicates that a specific threshold level of ARF is crucial for survival of the swa mutants.
Synthetic lethal interaction between arf1 and chc1:
The swa5-1 (chc1-5) mutant, which exhibits a ts growth phenotype and the most severe CPY transport defect of any swa mutant isolated, carries a mutation near the 3' end of the clathrin heavy chain (CHC1) gene. Here, we have shown that arf1 chc1-5 and [D26G]arf1 chc1-5 double mutants are inviable or exhibit an extreme growth defect not observed in the single mutants. The arf1 chc1-521 and arf1 chc1-57 double mutants are viable but grew extremely poorly. Together, these findings of strong genetic interactions between arf1 and chc1 mutations provide in vivo support for a functional interaction between ARF and clathrin. Because in vitro studies have suggested that ARF is required for the binding of AP-1, and subsequently clathrin, to Golgi membranes ( double mutant, because double mutants harboring ret1-1 (-COP) and chc1 have been constructed and show only a modest synthetic growth defect (data not shown).
It is not known if ARF provides a binding site for coat assembly on a membrane or if ARF alters the membrane in a manner that allows efficient coat assembly. ARF is a potent activator of mammalian phospholipase D and it appears that the requirement for ARF in COPI vesicle formation can be replaced by pretreatment of Golgi membrane with phospholipase D ( mutant ( mutant accumulates what appear to be large spheres of interconnected membrane tubules (
The role of clathrin in vacuolar protein transport:
In mammalian cells, the cytoplasmic domain of the mannose-6-phosphate receptors is recognized by clathrin/AP-1 at the trans-Golgi network and directly packaged into vesicles presumably targeted for prelysosomal (or endosomal) compartments (
A clathrin triskelion consists of three heavy chains and three light chains, in which the heavy chain C termini associate to form a vertex with radially extended arms. A core trimerization domain has been mapped by limited proteolysis and expression of recombinant fragments to residues 1550–1587 with flanking sequences up to amino acid 1615 of the mammalian heavy chain required for formation of stable trimers (57) has been shown to form trimers even at 37° (
The kinetics of CPY transport to the vacuole is not affected in chc1 null, chc1-57, or chc1-521 mutants (after 1 hr at 37°) but is significantly delayed in the chc1-5 mutant, which may imply that the Chc1-5 protein would have a dominant negative effect on protein transport. However, the CPY transport defect of chc1-5 mutants is clearly recessive (Figure 2C) and even overexpression of the chc1-5 allele in a wild-type strain does not affect CPY transport (data not shown). This suggests that hetero-trimeric clathrin coats consisting of wild-type and mutant heavy chains are functional but that the homo-trimeric Chc1-5 coat specifically interferes with CPY transport as the kinetics of ALP or invertase transport are relatively unaffected. The simplest explanation for these results is that the Chc1-5 protein can form coats on vesicles budding from the Golgi that contain CPY but the 28 missense amino acids at the C terminus partially interfere with the uncoating reaction, thereby interfering with the fusion of these vesicles with a prelysosomal compartment. Entrapment of ARF and clathrin on these vesicles could further reduce the available pool of these proteins in chc1-5 arf1 mutants and contribute to the synthetic lethal phenotype. Hsc70, an uncoating factor for clathrin, was found to bind near the vertex (C terminus) of isolated mammalian clathrin triskelia where it may be required to disrupt contacts between adjacent triskelia within a clathrin lattice (
An alternate explanation for the CPY transport defect in the chc1-5 mutant cannot be ruled out with the present evidence. Since late Golgi proteins are mislocalized to the plasma membrane of clathrin mutants, possibly including proteins of the transport machinery such as v-SNARES, the transport of CPY may be affected indirectly in the chc1-5 mutant. Although the Golgi protein mislocalization phenotype of chc1-5 cells appeared comparable to that of chc1-57 or chc1-521 cells, the chc1-5 mutant exhibited a glycosylation defect not observed in the other mutants. This suggests that the chc1-5 mutation perturbs Golgi organization more substantially than the other chc1 mutants and it is possible that the CPY transport route is more sensitive to perturbation of the Golgi than is the ALP transport route. Additional work will be required to distinguish between these two models.
| ACKNOWLEDGMENTS |
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We gratefully acknowledge Markus Aebi, Tim Stearns, Rick Kahn, Greg Payne, Sandra Lemmon, Bruce Horazdovsky, Catherine Jackson, Gerald Johnston, Alex Franzusoff, and Scott Emr for strains and plasmids and Sandra Lemmon for the Chc1p antibody. We also thank Jeff Flick and members in the Graham lab for comments on the manuscript and for helpful discussions. This work was supported by grants from the National Institutes of Health (GM-50409) and the National Science Foundation (MCB-9600835) to T.R.G. and a National Cancer Institute Center Grant (CA 68485).
Manuscript received March 5, 1998; Accepted for publication June 16, 1998.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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BEDNAREK, S. Y., M. RAVAZZOLA, M. HOSOBUCHI, M. AMHERDT, and A. PERRELET et al., 1995 COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast. Cell 83:1183-1196[Medline].
BENDER, A. and J. R. PRINGLE, 1991 Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:1295-1305
BOMAN, A. L., T. C. TAYLOR, P. MELANCON, and K. L. WILSON, 1992 A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature 358:512-514[Medline].
COWLES, C. R., G. ODORIZZI, G. S. PAYNE, and S. D. EMR, 1997 The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole. Cell 91:109-118[Medline].
DELL'ANGELICA, E. C., J. KLUMPERMAN, W. STOORVOGEL, and J. S. BONIFACINO, 1998 Association of the AP-3 adaptor complex with clathrin. Science 280:431-434
DONALDSON, J. G., D. FINAZZI, and R. D. KLAUSNER, 1992 Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360:350-352[Medline].
GAYNOR, E. C. and S. D. EMR, 1997 COPI-independent anterograde transport: cargo-selective ER to Golgi protein transport in yeast COPI mutants. J. Cell Biol. 136:789-802
GAYNOR, E. C., S. TE HEESEN, T. R. GRAHAM, M. AEBI, and S. D. EMR, 1994 Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol. 127:653-665
GAYNOR, E. C., C.-Y. CHEN, S. D. EMR, and T. R. GRAHAM, 1998 ARF is required for maintenance of yeast Golgi and endosome structure and function. Mol. Biol. Cell 9:653-670
GRAHAM, T. R. and V. A. KRASNOV, 1995 Sorting of yeast -1,3-mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein. Mol. Biol. Cell 6:809-824[Abstract].
GRAHAM, T. R., M. SEEGER, G. S. PAYNE, V. MACKAY, and S. D. EMR, 1994 Clathrin-dependent localization of 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J. Cell Biol. 127:667-678
HELMS, J. B. and J. E. ROTHMAN, 1992 Inhibition by brefeldin A of a Golgi membrane enzyme that catalyzes exchange of guanine nucleotide bound to ARF. Nature 360:352-354[Medline].
HEUSER, J. and C. J. STEER, 1989 Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction. J. Cell Biol. 109:1457-1466
HORAZDOVSKY, B. F., G. R. BUSCH, and S. D. EMR, 1994 VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO J. 13:1297-1309[Medline].
JOHNSON, L. M., V. A. BANKAITIS, and S. D. EMR, 1987 Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protein. Cell 48:875-885[Medline].