Copyright  1998 by the Genetics Society of America

An arf1⌬ Synthetic Lethal Screen Identifies a New Clathrin Heavy Chain Conditional Allele That Perturbs Vacuolar Protein Transport in Saccharomyces cerevisiae

Chih-Ying Chen and Todd R. Graham Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235 Manuscript received March 5, 1998 Accepted for publication June 16, 1998

ABSTRACT 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 3Ј 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 carboxypepti- dase 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.

ROTEIN transport between distinct organelles in clathrin (Simpson et al. 1996; Panek et al. 1997) al- Peukaryotic cells is carried out by membrane-bound though a recent study reported a direct interaction be- vesicles. Formation of transport vesicles involves assem- tween AP-3 and the clathrin heavy chain in vitro (Dell’ bly of cytosolic coat proteins onto the donor membrane, Angelica et al. 1998). selective packaging of the cargo proteins and finally Small GTP-binding proteins are required to initiate budding of the coated vesicles. Several types of vesicle (or prime) assembly of the coats. As Sar1p functions to coats that mediate different protein transport steps have recruit COPII to the ER membrane, ADP-ribosylation been studied in detail (reviewed in Schekman and Orci factor (ARF) appears to be required on the Golgi mem- 1996). The Sec23/24p and Sec13/31p complexes form brane to recruit COPI or AP-1/clathrin coats (Schek- COPII, which coats vesicles that bud from the endo- man and Orci 1996). The involvement of ARF in plasmic reticulum (ER). Coatomer, a heptameric (␣,␤,- clathrin/AP-1 recruitment was first suggested by the ␤Ј,␥,␦,ε,␨-COP) protein complex, coats COPI vesicles observation that AP-1 dissociated from Golgi mem- that mediate transport from the Golgi to the ER and branes of cells treated with brefeldin A (Robinson and between Golgi compartments. Two types of coat pro- Kreis 1992; Wong and Brodsky 1992), a drug that is teins contain clathrin, which is composed of heavy thought to specifically inhibit an ARF guanine nucleo- chains and light chains, associated with different adap- tide exchange factor (Donaldson et al. 1992; Helms tor protein (AP) complexes. In mammalian cells, clath- and Rothman 1992; Morinaga et al. 1996; Peyroche rin/AP-1 coated vesicles bud from the trans-Golgi net- et al. 1996). In vitro, the binding of AP-1 to Golgi mem- work and deliver cargo to endosomal compartments, branes and subsequent recruitment of clathrin is while clathrin/AP-2 drives endocytosis from the plasma dependent upon the GTP␥S-activated form of ARF membrane. Recently, a third adaptor complex (AP-3) (Stamnes and Rothman 1993; Traub et al. 1993). How- Simpson has been identified ( et al. 1996), which in yeast ever, GTP␥S-activated ARF can also mediate recruit- appears to mediate the delivery of proteins from the ment of AP-2 and COPI to endosomal membranes (Sea- Cowles trans-Golgi network directly to the vacuole ( et al. man et al. 1993; Whitney et al. 1995), and COPI to Piper Stepp 1997; et al. 1997; et al. 1997). This AP-3 com- ER membranes (Bednarek et al. 1995) and can inhibit plex has been suggested to function independently of endosome-endosome fusion (Lenhard et al. 1992) and nuclear envelope reassembly (Boman et al. 1992). The in vivo significance of these observations is still not clear. Corresponding author: Todd R. Graham, Department of Molecular Biology, Box 1820 Station B, Vanderbilt University, Nashville, TN In addition, treatment of cells with brefeldin A causes 37235. E-mail: [email protected] the dissociation of many proteins peripherally associ-

Genetics 150: 577–589 (October 1998) 578 C.-Y. Chen and T. R. Graham ated with the Golgi (Kooy et al. 1992; Podos et al. 1994; (Kranz and Holm 1990) with 6211 arf1⌬ (Gaynor et al. 1998), Misumi et al. 1997) and it is not known if this represents respectively. The swa5-1 mutant (CCY 2017) was backcrossed three times with a wild-type yeast strain (SEY6210) to produce a direct requirement for ARF in Golgi binding or is an CCY620-5. Yeast cells were grown on yeast extract, peptone, indirect consequence of perturbing Golgi structure and and dextrose (YPD), synthetic minimal (SD) media supple- function. mented as necessary (Sherman 1991). FOA (5Ј-fluoroorotic In yeast Saccharomyces cerevisiae, ARF is encoded by two acid; Sigma, St. Louis) media counterselective against growth ϩ Sikorski Boeke genes, ARF1 and ARF2, which encode proteins with 96% of Ura cells were prepared as described ( and 1991). Cells were grown overnight in liquid SD media con- identity that are probably redundant in function taining 0.2% yeast extract and required supplements for meta- (Stearns et al. 1990a). Double arf1⌬ arf2⌬ mutants are bolic labeling experiments. inviable, indicating that ARF is an essential protein in Plasmids pCC8218 and pCC616 were generated as follows: yeast. The Arf2 protein is only expressed at 10% of the a 1.8-kb EcoRI-PstI fragment carryingARF1 was subcloned from pRB1297 (Stearns et al. 1990a) into the polylinker of pPolyIII level of Arf1 protein, and strains carrying a deletion of Lathe kb-1.8ف Stearns ( et al. 1987) to produce pPolyIII-ARF1. An the ARF2 gene show a wild-type phenotype ( et NotI fragment from pPolyIII-ARF1 was inserted into pCH1153 -1.8ف al. 1990a,b). Strains harboring a deletion of ARF1 grow (Kranz and Holm 1990) to produce pCC8218 and an well, yet exhibit modest defects in protein secretion and kb PstI-BamHI fragment from pPolyIII-ARF1 was subcloned modification (Stearns et al. 1990a), and, perhaps more into pRS315 (Sikorski and Hieter 1989) to produce pCC616. significantly, arf1⌬ mutants exhibit a substantial alter- To produce pCC416, a 7.7-kb SalI-NruI fragment carrying CHC1 was subcloned from pCC1-2 (isolated from a genomic ation in the structure of Golgi and endosomal compart- library) into the SalI-SmaI sites of pRS416 (Sikorski and ments (Gaynor et al. 1998). This suggests that ARF plays Hieter 1989). a role not only in protein transport but also in organelle The isogenic chc1-ts strains were prepared by targeted inte- membrane dynamics (Gaynor et al. 1998). Strains har- gration of 5Ј truncated chc1 alleles into the CHC1 locus (de- Tan boring the arf1⌬ null mutation combined with muta- picted in Figure 7B). YIpchc521⌬Cla ( et al. 1993) was used to prepare the 6210 chc1-521 strain. To prepare pCC306 tions in RET1, SEC21, and SEC27, which respectively chc1-5, a 7.4-kb SalI-NotI fragment carrying chc1-5 was sub- encode ␣-, ␥-, and ␤Ј-COP subunits of the coatomer cloned from pCC416-1 into pRS306 (Sikorski and Hieter complex, were found to display a synthetic growth de- 1989). A 7.6-kb AatII-SalI fragment carrying chc1-⌬57 was sub- fect, whereas double mutants harboring arf1⌬ com- cloned from p⌬57 (Lemmon et al. 1991) into the SmaI-SalI bined with several other sec mutations do not exhibit sites of pRS306 to produce pCC306 chc1-⌬57. The 5Ј ends of Stearns Gaynor the chc1 genes were deleted by digesting the pCC306 plasmids synthetic growth defects ( et al. 1990b; with BglII and ClaI, blunt-ending and ligating to generate the et al. 1998). This provides in vivo evidence that ARF plays 3Ј chc1 series of plasmids. To prepare p 3Јchc1-⌬43, two PCR a specific role in coatomer function; however, there is primers were designed to delete the C-terminal 43 amino acids no such genetic evidence yet for ARF in the assembly of Chc1p: one is upstream of the last BamHI site in the CHC1 of AP-1/clathrin coats. open reading frame (5Ј-ACAAATTTGACCAATTGGGATTG- Ј To identify other proteins that functionally interact 3 ); the other introduces two stop codons and a SalI site after codon 1610 (5Ј-CGTCGAGTCGACTCATTATTTTTTTATGG with Arf1p, we have employed a genetic screen to search AGATTTCAAATGGC-3Ј). After PCR amplification, the prod- for the mutations that display synthetic lethality in com- ucts were digested with SalI and BamHI and subcloned into bination with the arf1⌬ null mutation and have identi- the SalI-BamHI sites of p 3Јchc1-5. To generate the 6210 chc1 fied seven complementation groups (SWA1-7 for syn- mutants, the p 3Јchc1 plasmids were linearized with AftII and thetically lethal with arf1⌬). In this study, we focused transformed into SEY6210 to target integration into the chro- mosomal CHC1 locus. Uraϩ transformants were tested for ts on characterization of SWA5, which was found to be growth. Total DNA was then isolated from ts transformants allelic to the clathrin heavy chain gene (CHC1). This and the correct integration event was confirmed by PCR and genetic interaction between arf1⌬ and chc1 provides in DNA sequencing. vivo support for a role for ARF in clathrin coat assembly. Isolation and characterization of swa mutants: To start the Surprisingly, chc1-5 (swa5-1) mutants exhibited mark- screen, CCY2011 and CCY 2804 were grown at 30Њ in liquid SD medium lacking uracil to stationary phase and treated with edly slow transport kinetics for carboxypeptidase Y 3% ethyl methanesulfonate (Fluka, St. Louis) for 30 min at .survival %80–60ف CPY) that was notobserved in three other chc1 tempera- 30Њ (Lawrence 1991), which resulted in) colonies per plate 500ف ture-sensitive (ts) mutants. The effect of chc1-5 on CPY Cells were plated on YPD to yield transport was much more substantial than the effect and incubated at 30Њ for 5–6 days. Uniformly red colonies were on invertase or alkaline phosphatase transport, further streaked twice onto YPD plates to confirm the nonsectoring phenotype and finally onto FOA medium to confirm plasmid- implicating clathrin in transport of CPY from the Golgi dependent growth. Mutant strains were crossed with the pa- to the vacuole. rental strain of opposite mating type and an ARF1 ARF2 ade2 ade3 strain, and the resulting diploids were replica-plated onto FOA medium to determine if mutations were recessive and if MATERIALS AND METHODS the nonsectoring phenotype resulted from plasmid integra- tion, respectively. All of the diploids were FOAR indicating Media, strains, and plasmid construction: Yeast strains and that the mutations were recessive and that none of the mutants plasmids used in this study are listed in Table 1. CCY 2011C recombined the URA3 and ADE3 genes into a chromosome. and CCY2804C are meiotic progeny from crosses of CH1305 For complementation analysis, mutants of opposite mating (Kranz and Holm 1990) with 6210 arf1⌬ and of CH1304 type were intercrossed and the resulting diploids were replica- arf1 Synthetic Lethality With chc1 579

TABLE 1 Yeast strains and plasmids used

Strain/plasmid Genotype Source Strain SEY6210 MAT␣ leu2-3,112 ura3-52 his3-⌬200 trp1-⌬901 lys2-801 suc2-⌬9 Robinson et al. (1988) 6210 arf1⌬ SEY6210 arf1⌬::HIS3 Gaynor et al. (1998) CCY 2011C MAT␣ leu2 ura3-52 his3 lys2-801 ade2 ade3 suc2-⌬9 arf1⌬::HIS3 This study CCY 2011 CCY2011C pCC8218 This study CCY 2013 CCY2011 swa1-2 (cs) This study CCY 2016 CCY2011 swa4-1 This study CCY 2017 CCY2011 swa5-1 (ts) This study CCY 2018 CCY2011 swa7-1 (cs) This study CCY 2804C MATa leu2 ura3-52 his3 trp1-⌬901 ade2 ade3 arf1⌬::HIS3 This study CCY 2804 CCY2804C pCC8218 This study CCY 2807 CCY2804 swa2-2 (cs) This study CCY 2808 CCY2804 swa3-2 (cs) This study CCY 2811 CCY2804 swa4-2 (cs) This study CCY620-5 MAT␣ leu2 ura3-52 his3 lys2-801 suc2-⌬9 chcl-5 This study 6210 chc1-5 SEY6210 chc1-5::URA3 This study 6210 chc1-⌬43 SEY6210 chc1-⌬43::URA3 This study 6210 chc1-⌬57 SEY6210 chc1-⌬57::URA3 This study 6210 chc1-521 SEY6210 chc1-521::URA3 This study GPY1103 MATa leu2-3, 112 ura3-52 his4-519 trp1 can1 chc1-⌬8::LEU2 Payne et al. (1987) GPY55-10B MAT␣ leu2-3,112 ura3-52 trp1 prb1 Payne and Schekman (1989) GPY396.1 MAT␣ leu2-3,112 ura3-52 trp1 prb1 chc1-521 Graham and Krasnov (1995) Plasmid pCC8218 YCp50-URA3 ADE3 ARF1 AmpR This study pCC616 pRS315-LEU2 ARF1 AmpR This study pCC315 pRS315-LEU2 CHC1 AmpR This study pCC416 pRS416-URA3 CHC1 AmpR This study pJCY1-58 pJCY1-35-URA3 [D26G]-arf1 AmpR Kahn et al. (1995) pJCY1-62 pJCY1-35URA3 [W66R]-arf1 AmpR Kahn et al. (1995) p3Јchc1-5 pRS306 chc1-5-⌬Cla::URA3 AmpR This study p3Јchc1-⌬43 pRS306 chc1-⌬43-⌬Cla::URA3 AmpR This study p3Јchc1-⌬57 pRS306 chc1-⌬57-⌬Cla::URA3 AmpR This study YIp chc521⌬Cla YIp5 chc1-521-⌬Cla::URA3 AmpR Tan et al. (1993) pM39S-304 pSEY304 (2␮ URA3) M39S (MNN1-SUC2 fusion) AmpR Graham and Krasnov (1995) pCYI-20-308 pSWYC308 (CEN4 ARS1 URA3) prc1::SUC2 AmpR Johnson et al. (1987)

plated onto FOA medium. To distinguish mutants that re- were tested for growth at 37Њ. Unassigned mutants have not quired ARF1 for survival from those that required ADE3 or yet been tested. URA3, mutants were transformed with pCC616 and the trans- To clone SWA5, a genomic library (Horazdovsky et al. formants were tested for growth on FOA and YPD media. 1994) was transformed into CCY620-5, and Leuϩ transform- FOAR or sectoring transformants were taken as ARF1-depen- ants were replica-plated onto selective medium and incubated dent. The results of the mutant screen are summarized as at 37Њ. Plasmid pCC1-2 isolated from a colony that grew at 37Њ follows: 63 nonsectoring mutants were isolated, among which was retransformed into CCY620-5 to confirm the ability to 3 were ARF1-independent, 16 were assigned to SWA1- SWA7, rescue the ts phenotype and was partially sequenced. 12 contained multiple mutations and were discarded, and 32 Cell labeling, immunoblotting, and invertase assay: Pulse- were set aside that could not clearly be assigned to a comple- chase metabolic labeling of cells with 35S amino acids and mentation group and did not exhibit a temperature condi- immunoprecipitations was done as previously described (Gay- tional growth defect. nor et al. 1994). To examine the secretion of invertase to the To test for complementation or suppression of swa mutants periplasmic space, CCY620-5 pCYI-20-308 transformants were by known genes, pEP1 (2␮ GCS1 LEU2 AmpR, a gift from converted to spheroplasts after pulse-chase labeling as pre- G. C. Johnston, Dalhousie University, Canada), pCLJ80 (2␮ viously described (Gaynorand Emr 1997). Half-times of trans- GEA1 LEU2 AmpR ; Peyroche et al. 1996), and AFB550 (CEN port were determined as previously described (Gaynor et al. SEC7 LEU2 AmpR , a gift from A. Franzusoff, University of 1998). For immunoblot analysis, yeast cultures were grown Colorado Health Sciences Center) were transformed into a overnight at 23Њ in YPD to mid-log phase. Ten OD600 of cells representative mutant from each complementation group and were collected, resuspended in 100 ␮l SDS-urea buffer (1% streaked on YPD plates to score the sectoring phenotype. In SDS; 6 m urea; 50 mm Tris-Cl, pH 7.5; and 1 mm EDTA), and addition, all of the swa ts mutants were crossed with strains lysed by vortexing with glass beads. Lysates containing 2 OD600 EGY1211-8c or EGY1211-14d (sec21-1, provided by S. Emr, equivalents were mixed with 4ϫ Laemmli sample buffer and University of California, San Diego) and the resultant strains subjected to SDS-PAGE. Western blots were probed with 580 C.-Y. Chen and T. R. Graham mouse monoclonal antibodies against CPY (Molecular Probes, Eugene, OR) and Chc1p (Lemmon et al. 1988), followed by incubation with horseradish peroxidase-conjugated goat anti- mouse antiserum (Jackson ImmunoResearch, West Grove, PA) and detection using Enhanced Chemiluminescence (Am- ersham, Arlington Heights, IN). Invertase assays were per- formed as previously described (Graham and Krasnov 1995).

RESULTS 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 (Koshland et al. 1985; Kranz and Holm 1990; Bender and Pringle 1991) was used to easily identify mutants that are able to live only when wild- type ARF1 is present. Strains that harbor arf1⌬ ade2 ade3 mutations (CCY2804C and CCY2011C) are white, while the transformants carrying wild-type ARF1 and ADE3 Figure 1.—Synthetic defects exhibited by arf1⌬ swa5-1 dou- genes linked together on a plasmid (pCC8218, URA3- ble mutants. (A) Wild type (CCY2011, arf1⌬ SWA5 pARF1) based) form red colonies that contain white sectors (Fig- and the swa5-1 mutant (CCY 2017, arf1⌬ swa5-1 pARF1)on ure 1A). This sectoring phenotype indicates the cells YPD medium at 30Њ form red/white sectoring and uniformly can readily lose the ARF1-ADE3-URA3 plasmid, which is red colonies, respectively. The nonsectoring phenotype of the ⌬ also demonstrated by the ability of these strains to grow mutant indicates that arf1 swa5-1 double mutants can sur- vive only when the plasmid (pCC8218) carrying ARF1-ADE3 on medium containing FOA, a drug that selects against is present. (B) Tetrad analysis of progeny derived from a colo- cross between wild-type and swa5-1 strains. The swa5-1 mutant 19,000ف cells harboring the URA3 gene. Among nies that survived ethyl methanesulfonate treatment, 63 (CCY2017) was mated with the parental wild-type strain mutant colonies consistently showed a nonsectoring red (CCY2011). The resultant diploids were sporulated, and mei- Њ phenotype on YPD medium (Figure 1A) and no growth otic segregants were incubated on YPD medium at 30 . Be- cause the ARF1-ADE3-URA3 plasmid (pCC8218) was lost fre- on FOA at 30Њ, indicating that these strains could not quently during meiosis, each tetrad yielded two robust (arf1⌬ survive without the plasmid. Three mutants were dis- SWA5) and two extremely slow-growing or inviable (arf1⌬ carded because they were unable to lose the original swa5-1) spore clones, indicating that the arf1⌬ synthetic ARF1-ADE3-URA3 plasmid when transformed with ARF1 growth is caused by a single gene mutation, swa5-1. carried on a LEU2-based vector (pCC616), suggesting the mutations that caused the nonsectoring phenotype were independent of Arf1p. mutants remained unassigned, including 27 that exhib- Because protein secretion is an essential process, mu- ited a conditional growth defect. tations in genes involved in the secretory pathway usually All of the strains displaying a conditional growth phe- affect the growth of the mutants. We screened for mu- notype were analyzed directly for protein transport de- tants that grew well at 30Њ. However, it was possible fects by pulse-chase experiments at their nonpermissive that these mutants would exhibit a conditional growth temperature and immunoprecipitation of the vacuolar defect. Because the nonsectoring mutants harbor a copy protein CPY (described in detail below). Ten of the 27 of wild-type ARF1 on a plasmid, additional phenotypes, unassigned conditional mutants displayed a defect in such as temperature conditional growth, should be at- CPY transport or glycosylation and were characterized tributable to the swa mutations. Therefore, all 60 of further to determine if this phenotype was caused by a the mutants were examined for cold sensitive (cs)or single gene mutation. The mutants were backcrossed temperature sensitive (ts) defects in growth at 15Њ or with the parental strain, diploids were sporulated, and 37Њ, respectively. Thirty-five of the mutants were either tetrads were dissected. During the tetrad analyses, we cs, ts, or both. found that the ARF1-ADE3-URA3 plasmid was frequently From backcrosses to parental strains it was deter- lost during meiosis and not recovered in the progeny. mined that all the mutants contained recessive muta- For example, instead of finding two sectoring vs. two tions (see materials and methods). Four complemen- nonsectoring progeny from a heterozygous diploid car- tation groups each containing two or more alleles were rying a single gene mutation, two viable (white, arf1⌬ defined (SWA1-SWA4, Figure 2A) by intercrossing the SWA) vs. two dead (arf1⌬ swa) progeny were usually mutants and testing for complementation of the non- observed (Figure 1B). Three more complementation sectoring phenotype in diploids. From this analysis, 47 groups were confirmed by the tetrad analyses, each con- arf1 Synthetic Lethality With chc1 581

phenotype, indicative of multiple mutations, and thus were not characterized further (see materials and of the predicted double %50ف ,methods). For swa5-1 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 con- ditional 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 (Stevens et al. 1982). Wild- type and mutant cells were grown to mid-log phase at permissive temperature (30Њ), shifted to the nonpermis- sive temperature (15Њ or 37Њ) for 1 hr, pulse-labeled with [35S]methionine for 10 min, and chased for 60 min (15Њ) or 15 min (37Њ). Aliquots of cells were removed at different chase times and CPY was recovered by immu- noprecipitation with anti-CPY antiserum. The half times of CPY transport from the ER to the vacuole were nearly min at 37Њ (Figure 5ف min at 15Њ (Figure 2B) and 60 2C and estimated from other experiments not shown) Figure 2.—Characterization of the swa mutants. (A) Seven in the wild-type cells. swa2-2, swa3-2, swa4-2, swa5-1, and complementation groups were defined by complementation swa7-1 mutants exhibited a defect in the kinetics of CPY tests and tetrad analyses. Strains tested were wild-type, CCY transport and a subtle defect in Golgi-specific glycosyla- 2011 or CCY 2804 (arf1⌬ SWA pARF1); arf1⌬, CCY 2011C or CCY 2804C (arf1⌬ SWA); and swa mutants (arf1⌬ swa pARF1) tion at their nonpermissive temperatures (Figure 2, B isolated from mutagenized cultures of the wild-type strains. and C). In several pulse-chase experiments with these Numbers of alleles displaying cs or ts phenotype are indicated mutants, we noticed that p2 CPY could not be resolved in parentheses. Fluoride sensitivity was tested on freshly pre- from p1 CPY and that mCPY migrated slightly faster in m pared YPD media containing 0, 20, 40, 60, or 80 m NaF. the gels than mCPY from wild-type cells. Figure 2A Strains tested for FϪ sensitivity were CCY2013 (swa1-2), CCY2807 (swa2-2), CCY 2808 (swa3-2), CCY 2016 (swa4-1), CCY 2017 shows a summary from at least two pulse-chase experi- (swa5-1), CCY 2018 (swa7-1), CCY 2011C and CCY2804C ments of the CPY transport kinetics observed in the swa (arf1⌬), and CCY 2011 and CCY 2804 (wild type). The numbers mutants at their nonpermissive temperatures, relative shown are the lowest concentrations of NaF on which the to wild-type and arf1⌬ cells. The swa3-2, swa4-2, swa6-1, mutants failed to grow. CPY transport was scored based on and swa7-1 mutants exhibited a defect in CPY trans- pulse-chase experiments as shown in B and C: ϩϩϩϩ indi- cates transport as efficient as wild type, ϩϩϩ is slightly (one- port comparable to that observed in arf1⌬ cells (ap- to twofold) slower than wild type, ϩϩ is as slow as the arf1⌬ proximately threefold delay in transport, marked as mutants (two- to threefold slower than wild type), and ϩ is ϩϩ). swa6-1 consistently incorporated significantly less more than threefold slower transport than wild type. (B and [35S]methionine into TCA precipitable proteins than C) Pulse-chase analysis of CPY transport in the swa mutants. the other mutants and is not shown in Figure 2. The Strains tested were CCY 2013 (swa1-2), CCY2807 (swa2-2), CCY 2808 (swa3-2), CCY 2811 (swa4-2), CCY 2017 (swa5-1), swa1-2 mutant was indistinguishable from wild type CCY 2018 (swa7-1), CCY 2804C (arf1⌬), and CCY2804 (WT). (ϩϩϩϩ), whereas swa2-2 mutants displayed a modest Cells were grown at 30Њ to mid-log phase. After shifting to a but reproducible transport defect (ϩϩϩ). The swa5-1 nonpermissive temperature (15Њ for cs,B;37Њfor ts, C) for mutant displayed the strongest defect in CPY transport, 1 hr, cells were labeled for 10 min and then chased for the with a three- to sixfold increase in the half time of indicated times and processed for immunoprecipitation with antiserum to CPY. CPY maturation (ϩ). For swa3-2, swa4-2, and swa5-1,a slight CPY transport defect was apparent at 30Њ but was exaggerated at the nonpermissive temperature. taining one mutant allele, and designated as SWA5- As described above, several of the swa mutants dis- SWA7. Seven of the temperature conditional mutants played cs growth and protein transport defects that are did not show 2:2 segregation of the synthetic lethal very similar to those observed in arf1⌬ cells (Stearns 582 C.-Y. Chen and T. R. Graham et al. 1990b). Moreover, we found that some mutants were also hypersensitive to fluoride, which is another phenotype exhibited by arf1⌬ mutants (Stearns et al. 1990b). The wild-type strains used in the study can grow on medium containing up to 80 mm NaF while the iso- genic arf1⌬ 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-gua- nine nucleotide exchange factor), and GCS1 (ARF- GTPase activating protein) were previously found to display genetic interactions with the arf1⌬ mutation (Stearns et al. 1990b; Peyroche et al. 1996; Poon et al. 1996). To test for allelism to the SWA genes defined here, plasmids carrying GEA1, GCS1, and SEC7 were transformed into at least one representative mutant from each SWA complementation group. However, none of the transformants showed a sectoring pheno- type, suggesting that these genes can neither suppress nor complement any of the swa mutants. In addition, all of the swa ts mutants were crossed with sec21 ts strains Figure 3.—CHC1 complements both ts growth and protein and the resultant diploids were all able to grow at 37Њ, transport defects of the swa5-1 mutant. (A) Single copy plas- suggesting that a sec21 mutant allele was not isolated in mids containing CDC68 and CHC1 (pCC1-2) or CHC1 alone this screen. (pCC315 CHC1) were able to complement the ts growth defect of a swa5-1 mutant (CCY620-5, ARF1 swa5-1). Deletion of Nsi I The clathrin heavy chain (CHC1) gene complements fragments in pCC1-2 disrupted the ability to complement the the ts and protein transport defect of swa5-1 mutants: ts phenotype. (B) Growth of CCY620-5 (swa5-1) pCC315 CHC1 Since the swa5-1 mutant exhibited a ts growth defect and CCY620-5 (swa5-1) pRS315 on SD minus leucine medium and markedly slow CPY transport kinetics, we decided incubated at the indicated temperatures. (C) SEY6210 (SWA5), to first focus on SWA5 characterization. The SWA5 gene CCY620-5 (swa5-1), CCY620-5 pCC315-CHC1 (swa5-1 pCHC1), and CCY620-5 pRS315 (swa5-1 vector only) were grown at 30Њ was cloned by complementing the ts phenotype of the to mid-log phase and converted to spheroplasts. After they mutant. From a yeast low-copy genomic library, we iso- were shifted to 37Њ for 1 hr, cells were labeled for 10 min and kb insert from chro- then chased for 0, 15, and 30 min. Cells and medium were-10.8ف lated a plasmid carrying an mosome VII (pCC1-2) containing two open reading separated by centrifugation and processed forimmunoprecipi- frames, CDC68 and CHC1 (Figure 3A). Deletion of a tation with antiserum to CPY. 1735-bp fragment in CHC1 (⌬NsiI) disrupted the ability of this plasmid to complement the growth defects of swa5-1 mutants at 37Њ while a fragment containing only swa5-1 mutant and the mutant carrying CHC1 on a plas- full-length CHC1 complemented the ts phenotype (Fig- mid. The swa5-1 mutant was again found to exhibit ure 3, A and B). Even though ARF has been implicated defects in CPY transport kinetics (approximately five- in AP-1/clathrin recruitment to Golgi membranes, this fold slower) and glycosylation of CPY, while wild-type was a surprising result because CPY transport has been CHC1 corrected both defects (Figure 3C). The stronger characterized in chc1 mutants previously with no defect transport defect observed in this experiment was most observed in a null mutant (Payne et al. 1988) and only likely the result of performing the pulse chase in sphero- a transient missorting defect observed in a chc1 ts (chc1- plasted cells. CPY was immunoprecipitated from both 521) strain shifted to the nonpermissive temperature cells and medium in this experiment, but very little (Seeger and Payne 1992a). Therefore, we tested to CPY was detected in the medium samples (Figure 3C), determine if CPY is missorted and secreted from the indicating that CPY was not being missorted. In addi- swa5-1 mutant and if CHC1 can correct the CPY trans- tion, all of the CPY was converted to the mature form port defects. Cells were converted to spheroplasts, and, at longer chase times (data not shown), indicating that after shifting to 37Њ for 1 hr, a pulse-chase experiment p2 CPY was not secreted from the cell. Shifting to 37Њ similar to that described above was performed with the for 5 min prior to labeling swa5-1 mutants caused a arf1 Synthetic Lethality With chc1 583

Figure 4.—The swa5-1 mutation maps near the 3Ј end of CHC1. Plasmids with the CHC1 open reading frame (pCC416) gapped at four different regions with the indicated restriction enzymes were trans- formed into CCY620-5 (swa5-1). Repaired plasmids were recovered from the yeast transformants and then amplified in Esche- richia coli and retransformed back into CCY620-5. A plasmid (pCC416-1) gap-re- paired at the very 3Ј end of CHC1 (gap 1) failed to complement the swa5-1 ts growth defect whereas those originally gapped else- where complemented. The dashed line in- dicates DNA copied from the CHC1 locus of the swa5-1 mutant. less dramatic CPY transport defect and still no CPY was and dropped substantially when the cells were shifted found in the medium (data not shown). Therefore, to 37Њ (Figure 5, lanes 4–6). Interestingly, the level of CPY is not missorted in the swa5-1 mutant, and CHC1 wild-type Chc1p also dropped after cells were shifted complemented both the ts growth and protein transport to 37Њ for 1 hr and increased somewhat by 2 hr (Fig- defects exhibited by swa5-1 mutants, suggesting that ure 5, lanes 1–3). The loss of mutant Chc1p after tem- swa5-1 may be an allele of CHC1. perature shift appeared more gradual for the chc1-521 The swa5-1 mutation resides within CHC1: To deter- mutant even compared to the isogenic wild-type strain mine whether SWA5 is allelic to CHC1, gap rescue (Figure 5, lanes 7–12). (Rothstein 1991) was performed to recover segments The genetic interaction between arf1 and chc1 is not of the CHC1 gene from the swa5-1 mutant. Plasmids allele specific: To test for a synthetic interaction of gapped at four different regions within CHC1 (Figure chc1-5 with arf1 mutations other than arf1⌬, two arf1 4) were transformed into a swa5-1 mutant to repair the mutant alleles carried on plasmids (Kahn et al. 1995) missing portions using sequence information from the were used: [D26G] arf1 (pJCY1-58) carrying a point chromosomal CHC1 gene of the swa5-1 mutant. The mutation in the GTP-binding domain, which encodes repaired plasmids were subsequently rescued, ampli- an Arf1p with a decreased affinity for GTP; and [W66R] fied, and transformed back into the swa5-1 mutants to arf1 (pJCY1-62), which is able to complement the fluo- test for the ability to complement the ts phenotype. The ride sensitivity of arf1⌬ mutants at 30Њ but not at 37Њ, rescued plasmid that was originally gapped at the very suggesting that the encoded Arf1p is partially defective. 3Ј end of CHC1 (Figure 4, Gap 1, ⌬AatII-BamHI) did Progeny of the cross between arf1⌬ pJCY1-58 (or pJCY1- not complement the swa5-1 ts growth defect, indicating 62) and chc1-5 mutants were characterized by random that a mutation(s) was located within this region. DNA spore analyses. The [W66R] arf1 allele can efficiently sequencing revealed that a G at nucleotide position rescue the synthetic lethality between chc1-5 and arf1⌬, 4831 is missing, which results in a frameshift mutation because an equal number of arf1⌬ chc1-5 and arf1⌬ 43 codons before the end of the open reading frame and SWA5 segregants were recovered among the progeny consequently a truncated clathrin heavy chain (Chc1p) that retained the plasmid. However, a modest growth 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 sta- bility of the mutant Chc1p. Strains harboring chc1-5, chc1-521,orchc1-⌬ alleles and wild-type strains were grown to mid-log phase at 23Њ and then shifted to 37Њ. Lysates from cells taken at the indicated time points were subjected to SDS-PAGE, blotted, and then probed Figure 5.—Stability of Chc1p at 37Њ in chc1 and wild-type with Chc1p and CPY antibodies, the latter to control CHC1 cells. Strains SEY6210 (CHC1, lanes 1–3), CCY620-5 for equal loading of the gel. As expected from the chc1-5 (chc1-5, lanes 4–6), GPY55-10B (CHC1, lanes 7–9), GPY396.1 mapping and sequencing data, the mobility of the mu- (chc1-521, lanes 9–12), and GPY1103 (chc1-⌬, lane 13) were grown at 23Њ and shifted to 37Њ for 0, 1, or 2 hr before lysis. tant protein was indistinguishable from that of the wild- Total cellular proteins were subjected toSDS-PAGE and immu- type Chc1p. However, the level of Chc1-5 protein was noblotted with monoclonal antibody against clathrin heavy reduced in cells growing at a permissive temperature chain (top) or CPY (bottom). 584 C.-Y. Chen and T. R. Graham defect was observed for the [W66R] arf1 chc1-5 double mutants at 30Њ 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 interac- tion 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 mu- tant 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 Figure plasma membrane: Previous studies showed that clath- 6.—Mislocalization of an Mnn1-invertase fusion pro- tein to the plasma membrane of the chc1-5 mutant. Strains rin function is required at late Golgi compartments for SEY6210 (WT) and CCY620-5 (chc1-5) harboring pM39S were retaining resident Golgi enzymes, like Mnn1p, Kex2p, grown at 23Њ to 0.5–1.0 OD600/ml. The cultures were shifted and dipeptidylaminopeptidase A, which are mislocal- to 37Њ and aliquots were removed at the indicated time points. ized to the plasma membrane of chc1 mutants (Payne The percentage of invertase at the cell surface was determined Graham Krasnov and Schekman 1989; Seeger and Payne 1992b; Gra- as previously described ( and 1995). ham et al. 1994). The loss of Kex2p from the late Golgi compartment results in inefficient processing of pro-␣- Payne factor and secretion of this precursor form from chc1 slow-growing viable cells in others ( et al. 1987; Lemmon mutants. We also found that the chc1-5 mutant (CCY620- et al. 1990). To determine if the defect in CPY 5) secreted ␣-factor precursor at the nonpermissive tem- transport kinetics exhibited by the chc1-5 mutant is due perature to the same extent as other chc1 mutants (data to the strain background or is an allele-specific effect, not shown). To directly determine if Golgi enzymes were we generated isogenic strains carrying chc1-5 and two mislocalized to the plasma membrane of the chc1-5 other well-studied chc1 ts mutant alleles (chc1-521 and Lemmon Seeger Payne mutant incubated at the nonpermissive temperature, chc1-⌬57; et al. 1991; and 1992b; perhaps causing the glycosylation defects (Figure 3C), Figure 7A) in the SEY6210 strain background and com- we analyzed the extent of mislocalization of the fusion pared the phenotypes. Integrating plasmids carrying protein M39I, containing the reporter enzyme invertase only the 3Ј half of the chc1 mutant alleles (5Ј⌬ chc1*) attached to the cytoplasmic tail and transmembrane were constructed, linearized, and transformed into domain (Golgi-localization signal) of Mnn1p. Wild-type SEY6210 to simultaneously disrupt the wild-type CHC1 and chc1-5 cells expressed 5–7% of M39I on the plasma gene and integrate the mutant alleles (Figure 7B). All membrane at the permissive temperature (Figure 6, 0 the resulting chc1 mutants were ts for growth. The iso- hr). In wild-type cells, the percentage of M39I in the genic strains were grown at 23Њ, shifted to 37Њ for 1 hr, plasma membrane did not change during a 2 hr incuba- and subjected to pulse-chase labeling to analyze CPY -transport. Mutants harboring chc1-521 and chc1-⌬57 ex -2.5ف tion at 37Њ. In contrast, chc1-5 cells mislocalized fold more M39I to the plasma membrane after tempera- hibited CPY transport kinetics similar to those of the ture shift (Figure 6). These phenotypes exhibited by wild-type strain, but the chc1-5 mutants still displayed the chc1-5 mutant, secretion of ␣-factor precursor and the slow transport defect (Figure 7C). These results mislocalization of a Golgi-retained reporter protein to indicate that the chc1-5 mutant allele indeed causes the the plasma membrane, are similar to those previously slow CPY transport and other chc1 ts alleles do not affect described for other chc1 mutants. CPY transport in the SEY6210 background after 1 hr The CPY transport defect exhibited by the chc1-5 mu- preincubation at 37Њ. tant is allele-specific: The chc1-5 mutant exhibited a Although chc1-5 and chc1-⌬57 encode similar mutant kinetic defect in CPY transport that has not been ob- forms of the clathrin heavy chain, the effects of these served in strains harboring other mutant alleles of chc1 mutations on CPY transport were quite different. To (Payne et al. 1988; Lemmon et al. 1991; Seeger and determine if this phenotypic difference was caused by Payne 1992a). We suspected that differences in strain the addition of 28 missense amino acids or by the loss background might affect the phenotype of the chc1 ts of the C-terminal 43 amino acids, we introduced a stop mutants. For instance, deletion of CHC1 results in a codon at position 1611 to produce the chc1-⌬43 allele lethal phenotype in some strain backgrounds, but causes (Figure 7A). The chc1-⌬43 mutation also causes a ts arf1 Synthetic Lethality With chc1 585

Figure 7.—CPY transport in isogenic chc1 ts mutants. (A) Schematic representation of the C termini of clathrin heavy chain encoded by the indicated chc1 alleles. The trimerization domain is indicated by the solid bar and underline and encompasses residues 1550–1615 in bovine Chc, which approximately correspond to residues 1556–1621 in yeast Chc1p. (B) Schematic representation depicting the construction of isogenic chc1 mutants. As described in detail in materials and methods, p3Јchc1 plasmids were linearized with AftII and transformed into SEY6210 to target integration into the CHC1 locus on chromosome VII. Wild-type CHC1 and the 3Ј end half of mutant chc1 were indicated by solid and gray arrows, respectively. The asterisk (*) indicates the region where the chc1 ts mutation resides. (C) CHC1, chc1-5, chc-⌬43, chc1-⌬57, and chc1-521 (isogenic strains in SEY6210 genetic background) were grown at 23Њ to mid-log phase. After shifting to 37Њ for 1 hr, cells were labeled for 10 min, then chased for 0, 15, 30, and 60 min, and processed for immunoprecipitation with antiserum to CPY. growth defect in the SEY6210 background, similar to perturbed in most vps mutants (Cowles et al. 1997; the other isogenic chc1 mutants generated. As shown Piper et al. 1997). To better define the protein trans- in Figure 7C, chc1-⌬43 mutant cells exhibited normal port defect in chc1-5 cells, we tested whether proteins transport kinetics for CPY but did exhibit a partial de- transported by the other two routes are affected as well. fect in glycosylation, as p2 CPY was not resolved well Pulse-chase experiments were performed to assess the from p1 CPY. Therefore, it appears that it is the 28 transport kinetics of invertase and ALP. The former is a missense amino acids at the very C terminus of the soluble protein that is rapidly secreted from cells and the Chc1-5 protein that interfere with transport, rather than latter is a vacuolar transmembrane protein that is appar- the lack of the C-terminal 43 amino acids. Furthermore, ently directly transported to the vacuole without passing the inefficient conversion of p1 to p2 CPY in the chc1-5 through an endosomal compartment. The ALP pathway mutant gives the impression that transport from the ER requires an adaptor-related AP-3 complex, which is to Golgi is the slow transport step. However, the chc1- thought to act independently of clathrin (Cowles et al. ⌬43 mutant exhibits a similar glycosylation defect but 1997; Piper et al. 1997; Panek et al. 1997; Stepp et al. normal transport kinetics for CPY, suggesting that the 1997; Vowels and Payne 1998). In wild-type cells la- apparent accumulation of p1 CPY in chc1-5 cells is beled at 37Њ, about half of the invertase was secreted by caused by underglycosylation rather than a defect in ER- 5 min of chase and nearly all was in the medium at 15 to-Golgi transport. This glycosylation defect is probably min (Figure 8A). The kinetics of invertase secretion secreted %90ف due to the mislocalization of ␣1,3 mannosyltransferase from chc1-5 cells was very similar, with (Mnn1p), which is primarily responsible for the conver- by 15 min. The invertase secreted from the chc1-5 mu- sion of p1 to p2 CPY (Graham et al. 1994). tant was clearly underglycosylated (Figure 8A), consis- chc1-5 specifically perturbs the vacuolar protein trans- tent with the underglycosylation of CPY and mislocaliza- port route taken by CPY: There are at least three known tion of Golgi enzymes described above (Figures 3C and routes for transfer of newly synthesized proteins out of 6). The kinetics of ALP transport was monitored by the the Golgi complex: (1) secretion to the cell surface; (2) time required for proteolytic processing of pro-ALP to transport via endocytic compartments (or endosomes) mature (m) ALP. In wild-type cells, slightly more than to the vacuole, which is the route taken by CPY and is half of the pro-ALP was processed at 5 min and nearly perturbed in vps mutants; and (3) the route taken by all was processed by 15 min (Figure 8B). Again, the alkaline phosphatase (ALP) to the vacuole, which is not kinetics of ALP transport in the chc1-5 cells was very 586 C.-Y. Chen and T. R. Graham

tions, 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 23Њ (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 comple- mentation groups defined here. This could be due to differences in the extent of the synthetic defect exhib- ited by distinct genetic strains. For instance, coatomer mutants were not found to be synthetically lethal with Figure 8.—Transport of invertase and alkaline phosphatase arf1⌬ in the SEY6210 background (Gaynor et al. 1998), in the chc1-5 mutant. (A) Strains SEY6210 pCYI-20 (WT) and indicating that differences in the genetic background CCY620-5 pCYI-20 (chc1-5) were labeled and then chased as may influence the extent of the synthetic interaction. described in the legend to Figure 7C and converted to sphero- plasts. Cells (C) and medium (M) were separated by centrifu- However, our screen was clearly not saturated and some gation and subjected to immunoprecipitation with antiserum of the original mutants that remain uncharacterized to invertase. (B) Isogenic strains SEY6210 (WT) and 6210 chc1- may carry mutant alleles of these genes. Because double 5 (chc1-5) were labeled and then chased as described in the arf1⌬ arf2⌬ mutants are inviable, ARF2 is a gene that legend to Figure 7C and subjected to immunoprecipitation should be identified in the screen. However, diploid with antiserum to ALP. 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 similar, with half of pro-ALP processed to mALP at 5 min ARF2 is insufficient for a diploid cell to live. Therefore, and most in the mature form at 15 min (Figure 8B). CPY strains carrying mutations in the ARF2 gene in this immunoprecipitated from the same extracts showed an screen would appear as harboring dominant mutations. approximately fourfold increase in the half time for Since all of the swa mutations tested were recessive, it transport (data not shown). In addition, the transport is unlikely that ARF2 is any of the SWA genes. This is kinetics of another vacuolar protein that follows the difficult to test directly because transformation of the CPY route, carboxypeptidase S (CPS), also showed a swa mutants with ARF2 carried on a CEN plasmid sup- three- to fourfold increase in the half time for vacuolar presses the nonsectoring phenotype of most of the mu- delivery (data not shown). These data suggest that pro- tants. The fact that a single copy of ARF2 rescues the tein transport through the secretory pathway is not sig- synthetic lethality between arf1⌬ and the swa mutations nificantly affected by the mutation, nor is trans- chc1-5 indicates that a specific threshold level of ARF is crucial port of ALP from the Golgi to the vacuole. Therefore, for survival of the swa mutants. the chc1-5 mutation specifically perturbs the transport Synthetic lethal interaction between arf1 and chc1: route taken by CPY and CPS from the Golgi to the The swa5-1 (chc1-5) mutant, which exhibits a ts growth vacuole, supporting a role for in this pathway. CHC1 phenotype and the most severe CPY transport defect of any swa mutant isolated, carries a mutation near the 3Ј end of the clathrin heavy chain (CHC1) gene. Here, DISCUSSION we have shown that arf1⌬ chc1-5 and [D26G]arf1 chc1-5 Identification of mutations that exhibit synthetic le- double mutants are inviable or exhibit an extreme thality with arf1⌬: We have initiated a genetic screen to growth defect not observed in the single mutants. The find factors that functionally interact with ARF and have arf1⌬ chc1-521 and arf1⌬ chc1-⌬57 double mutants are defined seven complementation groups of swa mutants. viable but grew extremely poorly. Together, these find- Strains harboring the arf1⌬ null allele exhibit pheno- ings of strong genetic interactions between arf1 and types of cs growth, hypersensitivity to fluoride ions, an chc1 mutations provide in vivo support for a functional approximately threefold delay in the kinetics of protein interaction between ARF and clathrin. Because in vitro transport through the secretory pathway, and Golgi- studies have suggested that ARF is required for the bind- specific glycosylation defects (Stearns et al. 1990a,b; ing of AP-1, and subsequently clathrin, to Golgi mem- Gaynor et al. 1998). Importantly, most of the swa mu- branes (Stamnes and Rothman 1993; Traub et al. tants exhibit very similar phenotypes suggesting that 1993), perhaps the combination of a decreased concen- these mutations perturb cellular functions similar to tration of ARF and clathrin heavy chain in the double those perturbed in the arf1⌬ mutant. The basis of the mutants reduces the membrane-associated clathrin be- screen, synthetic lethality between swa and arf1⌬ muta- low a threshold required for our strains to survive. It is arf1 Synthetic Lethality With chc1 587 less likely that a requirement for ARF in the early secre- requires the adaptor complex AP-3. Our data support tory pathway combined with one for clathrin in the later the view that AP-3 functions independently of clathrin secretory pathway causes synthetic lethality of the chc1 because ALP transport is unaffected in the chc1-5 mu- arf1⌬ double mutant, because double mutants harbor- tant. ing ret1-1 (␣-COP) and chc1 have been constructed and A clathrin triskelion consists of three heavy chains and show only a modest synthetic growth defect (data not three light chains, in which the heavy chain C termini shown). associate to form a vertex with radially extended arms. A It is not known if ARF provides a binding site for coat core trimerization domain has been mapped by limited assembly on a membrane or if ARF alters the membrane proteolysis and expression of recombinant fragments in a manner that allows efficient coat assembly. ARF is to residues 1550–1587 with flanking sequences up to a potent activator of mammalian phospholipase D and amino acid 1615 of the mammalian heavy chain re- it appears that the requirement for ARF in COPI vesicle quired for formation of stable trimers (Liu et al. 1995). formation can be replaced by pretreatment of Golgi The chc1-5 frameshift mutation lies within the region membrane with phospholipase D (Ktistakis et al. flanking the core trimerization domain (corresponding 1996). In this regard, it is significant that we have pre- to amino acid 1605 of the mammalian heavy chain), viously observed striking morphological changes in the which may cause triskelia to become unstable. In fact, structure of Golgi and endosomes in the arf1⌬ mutant a substantial reduction in the amount of Chc1p in the (Gaynor et al. 1998), which are distinct from the struc- chc1-5 mutant was observed, particularly at 37Њ (Figure tures observed in clathrin or coatomer mutants. The 5). It is likely that the remaining Chc1-5 protein can arf1⌬ mutant accumulates what appear to be large oligomerize because the chc1-5 mutant does not exhibit spheres of interconnected membrane tubules (Gaynor phenotypes suggesting a clathrin deficiency at a permis- et al. 1998). It is possible that these structures result sive temperature and a similar mutant heavy chain from an altered membrane composition and do not (Chc1-⌬57) has been shown to form trimers even at 37Њ provide the appropriate surface for clathrin to assemble (Lemmon et al. 1991). on or bud vesicles from. This may provide an alternative The kinetics of CPY transport to the vacuole is not explanation for the observed synthetic lethality. affected in chc1 null, chc1-⌬57,orchc1-521 mutants (after The role of clathrin in vacuolar protein transport: In 1 hr at 37Њ) but is significantly delayed in the chc1-5 mammalian cells, the cytoplasmic domain of the man- mutant, which may imply that the Chc1-5 protein would nose-6-phosphate receptors is recognized by clathrin/ have a dominant negative effect on protein transport. AP-1 at the trans-Golgi network and directly packaged However, the CPY transport defect of chc1-5 mutants is into vesicles presumably targeted for prelysosomal (or clearly recessive (Figure 2C) and even overexpression endosomal) compartments (Traub and Kornfeld of the chc1-5 allele in a wild-type strain does not affect 1997). In yeast, clathrin’s role in the Golgi complex to CPY transport (data not shown). This suggests that het- sort vacuolar proteins from the secretory pathway is still ero-trimeric clathrin coats consisting of wild-type and controversial. Strains harboring a deletion of CHC1 do mutant heavy chains are functional but that the homo- not show any protein transport or sorting defects trimeric Chc1-5 coat specifically interferes with CPY (Payne et al. 1988), suggesting that clathrin is not in- transport as the kinetics of ALP or invertase transport volved in vacuolar protein sorting. On the other hand, are relatively unaffected. The simplest explanation for the finding that strains harboring chc1-521 transiently these results is that the Chc1-5 protein can form coats secrete precursors to soluble vacuolar proteins, such as on vesicles budding from the Golgi that contain CPY CPY, upon shifting to the nonpermissive temperature but the 28 missense amino acids at the C terminus par- provides the only evidence of a role for clathrin in sort- tially interfere with the uncoating reaction, thereby in- ing vacuolar proteins (Seeger and Payne 1992a). After terfering with the fusion of these vesicles with a prelyso- extended incubation at 37Њ chc1-521 cells correct the somal compartment. Entrapment of ARF and clathrin missorting defect and restore CPY sorting, presumably on these vesicles could further reduce the available pool through a clathrin-independent pathway (Seeger and of these proteins in chc1-5 arf1⌬ mutants and contribute Payne 1992a). Strains harboring the chc1-5 mutant al- to the synthetic lethal phenotype. Hsc70, an uncoating lele isolated in this study exhibited a striking delay in factor for clathrin, was found to bind near the vertex CPY and CPS transport but only a slight effect on the (C terminus) of isolated mammalian clathrin triskelia transport kinetics of ALP and invertase. These data sup- where it may be required to disrupt contacts between port a role for clathrin in the transport pathway that adjacent triskelia within a clathrin lattice (Heuser and delivers CPY to the vacuole, which may be analogous to Steer 1989). Perhaps this reaction is inefficient in the route taken by mammalian lysosomal enzymes that strains harboring chc1-5 mutation. bear mannose-6-phosphate. As previously suggested, it An alternate explanation for the CPY transport defect is possible that clathrin is essential for normal CPY trans- in the chc1-5 mutant cannot be ruled out with the pres- port but clathrin mutants adapt by diverting CPY into ent evidence. Since late Golgi proteins are mislocalized the ALP pathway (Seeger and Payne 1992a), which to the plasma membrane of clathrin mutants, possibly 588 C.-Y. Chen and T. R. Graham including proteins of the transport machinery such as intermediate in the vesicle uncoating reaction. J. Cell Biol. 109: 1457–1466. v-SNARES, the transport of CPY may be affected indi- Horazdovsky, B. F., G. R. Busch and S. D. Emr, 1994 VPS21 en- rectly in the chc1-5 mutant. Although the Golgi protein codes a rab5-like GTP binding protein that is required for the mislocalization phenotype of chc1-5 cells appeared com- sorting of yeast vacuolar proteins. EMBO J. 13: 1297–1309. Johnson, L. M., V. A. Bankaitis and S. D. Emr, 1987 Distinct se- parable to that of chc1-⌬57 or chc1-521 cells, the chc1-5 quence determinants direct intracellular sorting and modifica- mutant exhibited a glycosylation defect not observed in tion of a yeast vacuolar protein. Cell 48: 875–885. the other mutants. This suggests that the chc1-5 mutation Kahn, R. A., J. Clark, C. Rulka, T. Stearns, C. J. Zhang et al., 1995 Mutational analysis of Saccharomyces cerevisiae ARF1. J. Biol. Chem. perturbs Golgi organization more substantially than the 270: 143–150. other chc1 mutants and it is possible that the CPY trans- Kooy, J., B. H. Toh, J. M. Pettitt, R. Erlich and P. A. Gleeson, port route is more sensitive to perturbation of the Golgi 1992 Human autoantibodies as reagents to conserved Golgi components: characterization of a peripheral, 230-kDa compart- than is the ALP transport route. Additional work will ment-specific Golgi protein. J. Biol. Chem. 267: 20255–20263. be required to distinguish between these two models. 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