Copyright 1998 by the Genetics Society of America An arf1D Synthetic Lethal Screen Identi®es 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 arf1D allele and de®ned seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1D). Most of the swa mutants exhibit phenotypes comparable to arf1D mutants such as temperature-conditional growth, hypersensitivity to ¯uoride 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 39 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 signi®cant 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 signi®cantly affected in the chc1-5 mutant, further implicating clathrin speci®cally 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 ®nally 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 (a,b,- clathrin/AP-1 recruitment was ®rst suggested by the b9,g,d,ε,z-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 speci®cally 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 GTPgS-activated form of ARF membrane. Recently, a third adaptor complex (AP-3) (Stamnes and Rothman 1993; Traub et al. 1993). How- Simpson has been identi®ed ( et al. 1996), which in yeast ever, GTPgS-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 signi®cance 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 arf1D (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 (59-¯uoroorotic In yeast Saccharomyces cerevisiae, ARF is encoded by two acid; Sigma, St. Louis) media counterselective against growth 1 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 arf1D arf2D 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 Stearns ( et al. 1987) to produce pPolyIII-ARF1. An z1.8-kb the ARF2 gene show a wild-type phenotype ( et NotI fragment from pPolyIII-ARF1 was inserted into pCH1153 al. 1990a,b). Strains harboring a deletion of ARF1 grow (Kranz and Holm 1990) to produce pCC8218 and an z1.8- well, yet exhibit modest defects in protein secretion and kb PstI-BamHI fragment from pPolyIII-ARF1 was subcloned modi®cation (Stearns et al. 1990a), and, perhaps more into pRS315 (Sikorski and Hieter 1989) to produce pCC616. signi®cantly, arf1D 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 59 truncated chc1 alleles into the CHC1 locus (de- Tan boring the arf1D null mutation combined with muta- picted in Figure 7B). YIpchc521DCla ( 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 a-, g-, and b9-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-D57 was sub- fect, whereas double mutants harboring arf1D com- cloned from pD57 (Lemmon et al. 1991) into the SmaI-SalI bined with several other sec mutations do not exhibit sites of pRS306 to produce pCC306 chc1-D57. The 59 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 39 chc1 series of plasmids. To prepare p 39chc1-D43, two PCR a speci®c 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 (59-ACAAATTTGACCAATTGGGATTG- 9 To identify other proteins that functionally interact 3 ); the other introduces two stop codons and a SalI site after codon 1610 (59-CGTCGAGTCGACTCATTATTTTTTTATGG with Arf1p, we have employed a genetic screen to search AGATTTCAAATGGC-39). After PCR ampli®cation, the prod- for the mutations that display synthetic lethality in com- ucts were digested with SalI and BamHI and subcloned into bination with the arf1D null mutation and have identi- the SalI-BamHI sites of p 39chc1-5. To generate the 6210 chc1 ®ed seven complementation groups (SWA1-7 for syn- mutants, the p 39chc1 plasmids were linearized with AftII and thetically lethal with arf1D). In this study, we focused transformed into SEY6210 to target integration into the chro- mosomal CHC1 locus. Ura1 transformants were tested for ts on characterization of SWA5, which was found to be growth.