Four mutants defective in endocytosis were isolated by screening a collection of temper- ature-sensitive yeast mutants. Three mutations define new END genes: ...
Molecular Biology of the Cell Vol. 6, 1721-1742, December 1995
end5, end6, and end7: Mutations that Cause Actin Delocalization and Block the Internalization Step of Endocytosis in Saccharomyces cerevisiae Alan L. Munn, Brian J. Stevenson, M. Isabel Geli, and Howard Riezman* Department of Biochemistry, Biozentrum of the University of Basel, Basel, CH-4056, Switzerland Submitted May 17, 1995; Accepted September 27, 1995 Monitoring Editor: David Botstein
Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACTI, respectively. VRP1 and RVS161 are required for correct actin localization and ACTi encodes actin. To our surprise, the end6 -1 mutation fails to complement the actl-l mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrplp, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane. INTRODUCTION The yeast Saccharomyces cerevisiae has an endocytic pathway for the internalization of cell surface receptor/ligand complexes and for the removal of other surface proteins (reviewed in Riezman, 1993). Internalization of the yeast mating pheromone a-factor, bound to the a-factor receptor (STE2 gene product), can be assayed using radiolabeled a-factor and was found to be time, temperature, and energy dependent (Chvatchko et al., 1986; Dulic et al., 1991). Other work has shown that pheromone uptake is accompanied by uptake of the receptor (Jenness and Spatrick, 1986; Davis et al., 1993; Tan et al., 1993; Schandel and Jenness, 1994). Sequences within the cytoplasmic tail of the a-factor receptor, notably the DAKSS sequence, are important in promoting internalization (Rohrer et al., 1993). a-Factor uptake is followed by passage of the a-factor through at least two internal membrane* Corresponding author. Abbreviations used: CPY, carboxypeptidase Y; LY, lucifer yellow; PRR, proline-rich regions; Ts, temperature-sensitive.
© 1995 by The American Society for Cell Biology
bounded compartments with distinct densities before delivery to the vacuole, where the pheromone is degraded by resident proteases (Singer and Riezman, 1990; Singer-Kruger et al., 1993). Recent work has led to the discovery that the actin cytoskeleton plays an essential role in the internalization step of endocytosis in yeast (Kubler and Riezman, 1993). actl-l and actl-2 mutants, which express altered forms of actin, are blocked in internalization of a-factor by receptor-mediated endocytosis at the restrictive temperature. This defect is rapidly imposed at the restrictive temperature in the actl-l mutant, suggesting that the requirement for actin is direct. Mutants that are deficient in fimbrin due to a disruption of the SAC6 gene (sac6::URA3) are also blocked at the internalization step of endocytosis (Kubler and Riezman, 1993); however, not all mutations that affect the actin cytoskeleton affect a-factor uptake. Mutants defective in myosin type V (myo2-66), for example, display altered actin localization and inviability at the restrictive temperature (Johnston et al., 1991), but internalize a-factor with normal kinetics (Kubler et al., 1721
A.L. Munn et al.
1994). In addition, the 85-kDa actin-binding protein encoded by the ABP1 gene is not required either for actin localization or for endocytosis (Drubin et al., 1988; Adams et al., 1993; Kubler and Riezman, 1993). In S. cerevisiae, actin filaments occur in two forms that can be distinguished by light microscopy (Adams and Pringle, 1984). Actin cables extend through the cytoplasm of the cell and have a filamentous appearance when viewed by immunofluorescence, while actin cortical patches are found at the cell surface and have a distinct punctate appearance. It has been proposed that cortical actin patches provide osmotic support during the insertion of new cell wall material, however, the exact physiological roles of the cortical patches and cables are not yet clear (Mulholland et al., 1994). During the cell cycle, the filamentous actin distribution changes (Kilmartin and Adams, 1984). In cells with small buds the patches are concentrated in the bud, but in cells with large buds the patches are randomly distributed. In actl-l and actl-2 mutants, cortical actin patches are evenly distributed over the mother and daughter cell surfaces at all stages of the cell cycle (Novick and Botstein, 1985). A number of actin-binding proteins have been identified in yeast, including profilin (Haarer et al., 1990), cofilin (Moon et al., 1993), capping protein (Amatruda and Cooper, 1992), an 85-kDa actin-binding protein (Drubin et al., 1988), two tropomyosins (Liu and Bretscher, 1989a; Drees et al., 1995), fimbrin (Drubin et al., 1988; Adams et al., 1989,1991), and several myosins (Johnston et al., 1991; Goodson and Spudich, 1993) (see review by Welch et al., 1994). The 85-kDa actin-binding protein is localized only to cortical patches (Drubin et al., 1988), while other cytoskeletal proteins are localized only to cables, e.g. tropomyosin 1 (Liu and Bretscher, 1989b), or to both types of structure, e.g. fimbrin (Drubin et al., 1988; Adams et al., 1989, 1991). Mutations affecting many of these proteins (e.g. profilin, capping protein, fimbrin, and myosin type V) lead to a delocalization of cortical actin patches as do the actl-l and actl-2 mutations (Novick and Botstein, 1985; Haarer et al., 1990; Adams et al., 1991; Johnston et al., 1991; Amatruda et al., 1992). In addition to components of the actin cytoskeleton, clathrin heavy chain (CHC1 gene product) is also required for internalization of a-factor with wild-type kinetics (Payne et al., 1988). The involvement seems to be direct, because the chcl-521 mutant, which is temperature-sensitive for function, is blocked for a-factor uptake immediately upon shift to the restrictive temperature (Tan et al., 1993). Calmodulin also plays a role in the internalization step of endocytosis, because a temperature-sensitive calmodulin mutant (cmdl-1) is unable to internalize a-factor at the restrictive temperature and the effect of this mutation is also rapidly installed after temperature shift (Kubler et al., 1994). This mutant exhibits delocalized cortical actin patches 1722
at the restrictive temperature like mutants affected in actin and various actin binding proteins (Brockerhoff and Davis, 1992). We have initiated a genetic approach to identify proteins that participate in the receptor-mediated endocytosis of a-factor. By screening a collection of temperature-sensitive mutants for defects in uptake of a-factor, the end3-1 and end4-1 mutants were obtained (Raths et al., 1993). Both mutants are temperature sensitive for growth and have an altered actin staining pattern (Raths et al., 1993; Benedetti et al., 1994; Wesp, unpublished observation), but delivery of soluble vacuolar hydrolases to the vacuole is normal and the vacuole has normal morphology as judged by light microscopy (Raths et al., 1993). The END3 gene product is predicted to be a soluble 40-kDa protein with putative Ca2+- and PIP2-binding sites (Benedetti et al., 1994). Sequencing of the END4 gene showed that it is identical to the SLA2 gene identified by Holtzman et al. (1993) (Hicke, unpublished results). SLA2 encodes a 109-kDa protein that localizes to cortical actin patches and that exhibits amino acid sequence homology at its carboxyterminus to the mammalian focal adhesion site protein, talin (Holtzman et al., 1993; Yang and Drubin, cited in Li et al., 1995). In the endocytic pathway, the END3 and END4 gene products are directly required for a-factor internalization at the plasma membrane and not for subsequent transport to the vacuole (Raths et al., 1993; Benedetti et al., 1994). Here we report the isolation and characterization of four new mutants that are defective in a-factor internalization. These mutants were obtained by preparing a collection of mutants that are temperature sensitive for growth and then screening each member of the collection with a rapid a-factor uptake assay. Three mutants carry mutations in genes not previously identified in our screens (END5, END6, and END7), while the fourth mutant carries a new allele of END4 (end42). Interestingly, all of the new mutants have defects in cytoskeletal organization, like the end3-1 and end4-1 mutants isolated by Raths et al. (1993). Phenotypic characterization of the mutants and cloning of the genes has provided new insight into the subset of components of the actin-based cytoskeleton that is involved in endocytosis. MATERIALS AND METHODS Media and Strains The yeast strains used in this work are described in Table 1A. YPUAD-rich medium and SD selective medium were as described (Dulic et al., 1991) except that YPUAD contained 40 ,ug/ml adenine and uracil. Where necessary, SD was supplemented to 40 ,ug/ml with one or more of the following nutrients: adenine, uracil, tryptophan, leucine, histidine, tyrosine, and lysine. The nitrogen-limiting medium containing erythrosine B that we used was NO.05/ erythrosine B medium as described by Crouzet et al. (1991). YPUAD, SD, and NO.05/erythrosine B solid media contained 2% Bactoagar Molecular Biology of the Cell
Mutations Blocking Endocytosis
Table 1A. Yeast strains Name
Genotype
Source
RH144-3A RH144-3D RH977 RH2063 RH2066 RH2068 RH2069 RH2071
Lab. strain Lab. strain Lab. strain A. Goffeau This study This study This study This study
RH2073 RH2075 RH2077 RH2079 RH2080 RH2082 RH2161 RH2426 RH2514 RH2515 RH2516
MATa his4 leu2 ura3 barl MATa his4 leu2 ura3 barl MATa his4 leu2 ura3 trpl::URA3 barl MATa his3 leu2 ura3 trpl ade2 adpl::URA3 MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/TRPI end5-1/end5-1 barl/barl MATa his4 leu2 ura3 end7-1 barl MATa his4 leu2 ura3 end7-1 barl MATa/MATa his4/HIS4 his3/HIS3 leu2/leu2 ura3/ura3 trplITRPl ade2/ADE2 adpl::URA3/ADPI end61/END6 barlI/BARI MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/TRPI end6-1/END6 barl/barl MATa his4 leu2 ura3 end5-1 barl MATa his4 leu2 ura3 end5-1 barl MATa his4 leu2 ura3 end6-1 barl MATa his4 leu2 ura3 trpl::URA3 end6-1 barl MATa his4 leu2 ura3 end6-1 barl MATa/MATa his4/his4 leu2/leu2 ura3/URA3 lys2/LYS2 actl-l/actl-l barl/barl MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/trpl::URA3 barl/barl MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/TRPI lys2/LYS2 actl-i/ACTI barl/barl MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/TRPI end6-1/end6-1 barl/bar MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/TRP1 lys2/LYS2 actl-iIACTi end6-1/END6
RH2643 RH2950 RH2951 RH3118 RH3119 RH3120 RH3121 LG88-7B LG430-1A PHY102
barl/barl MATa/MATa his4/his4 leu2/leu2 ura3/ura3 trpl::URA3/TRP1 end7-1/end7-1 barl/barl MATa his4 leu2 ura3 trpl::URA3 rvsl67::TRP1 barl MATa his4 leu2 ura3 trpl::URA3 rvsl67::TRPI barl MATa/MATa his4/HIS4 ura3/ura3 trpl::URA3/TRPI rvs16l(end6)A/rvsl6l(end6)A barl/barl MATa/MATa his4/his4 leu2/LEU2 ura3/ura3 trpl::URA3/TRPI rvsl61(end6)A/RVS161(END6) barl/barl MATa/MATa his4/his4 leu2/LEU2 ura3/URA3 rvsl6i(end6)A/RVS161(END6) acti/ACTi barl/barl MATa/MATa his4/his4 leu2/LEU2 ura3/ura3 trpl::URA3/TRPI rvsl6I(end6)A/end6-1 barl/barl MATa his4 ura3 rvsl6i(end6)A barl MATa trpl rvsl67::TRPI MATa leu2 ura3 his3 trpl Iys2 suc2 GAL vps34Al::TRP1
(Difco, Detroit, MI). SD/YE was SD enriched with 0.2% yeast extract (Difco). Bacterial strains used for plasmid maintenance and amplification were XL1-blue, JM109, and DH5a. Their genotype has been described previously (Sambrook et al., 1989). Bacterial strains were grown in LB-rich medium prepared as described by Sambrook et al. (1989). Ampicillin (Boehringer Mannheim [Schweiz] AG, Rotkreuz, Switzerland), where needed for plasmid selection, was added at 100 ,ug/ml. Solid medium contained 2% Bactoagar (Difco). The M13 helper phage used for preparation of single-stranded pBSKII/PBKSII plasmid DNA was K07 (Vieira and Messing, 1987). Table 1B lists the plasmids used in this study.
This study This study This study This study This study This study Lab. strain This study This study This study This study This study This study This study This study This study This study This study A. Breton Bauer et al., 1993 Herman and Emr, 1990
Lucifer Yellow carbohydrazide (LY) was obtained from Fluka (Buchs, Switzerland). Rhodamine-labeled phalloidin was obtained from Sigma Chemical (St. Louis, MO). 35S a-factor was prepared as described (Dulic et al., 1991) with slight modification (Munn and Riezman, 1994). [35S]Methionine, [35S]cysteine used in pulse-chase metabolic labeling experiments was obtained from New England Nuclear (Boston, MA). Ethylmethanesulfonic acid was obtained from Sigma Chemical. Restriction enzymes, DNA polymerases, and DNA ligase were purchased from Boehringer Mannheim (Switzerland) AG, New England Biolabs (Beverly, MA), United States Bio-
Table 1B. Plasmids used in this work
Plasmid
Description
Source
pUBl-3 pEND5.1 pSRl pSR2 pT7BLUE pT7end7-1 YCplac33 YCplaclll pFL38
RVS161/END6 gene on a 2.2-kb EcoRI fragment in pFL38 (URA3) END5 on a 5.6-kb genomic insert in YCplaclll END3 gene on a -4-kb fragment from partial Sau3A digest in pSEY8 END4 gene on a -16-kb fragment from partial Sau3A digest in YCp5O Vector for cloning PCR fragments pT7BLUE containing PCR fragment with the end7-1 mutant gene Yeast centromere vector (URA3) Yeast centromere vector (LEU2) Yeast centromere vector (URA3) Yeast centromere vector (URA3)
Crouzet et al., 1991 This study Lab. plasmid Lab. plasmid Novagen This study Gietz and Sugino, 1988 Gietz and Sugino, 1988 Bonneaud et al., 1991 Rose et al., 1987
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chemical (Cleveland, OH), or Stratagene Cloning Systems (La Jolla, CA) and used as specified by the manufacturer. Polymerase chain reaction (PCR) was performed with SuperTaq polymerase from P.H. Stehelin and Cie AG (Basel, Switzerland). Oligonucleotide primers for PCR were made on an Applied Biosystems 380B Oligonucleotide Synthesizer (Applied Biosystems, Foster City, CA). DNA sequencing was performed with a United States Biochemical Sequenase II DNA sequencing kit. Sequencing reactions were subjected to electrophoresis using a Sequi-Gen DNA Sequencing Cell from Bio-Rad (Hercules, CA). PCR was carried out in a TRIO-Thermoblock obtained from Biometra GmbH (Gottingen, Germany). Autoradiography was performed with XAR x-ray film obtained from Kodak (Rochester, NY), and the films were developed with a Fuji automatic film processor.
Yeast Genetic
Techniques
Mating of haploid strains of yeast, sporulation of MATa/MATa diploid strains, and tetrad analysis were performed as described in Sherman et al. (1974). Transformation of yeast with plasmid DNA was accomplished by a modification of the method of Gietz et al. (1992). Cells were grown in YPUAD (100 ml) at 24°C to a density of 107 cells/ml. The cells were then harvested, washed in 15 ml of 100 mM lithium acetate/TE at pH 7.5, and resuspended in 1 ml of 100 mM lithium acetate/TE. One hundred microliters of cells were then mixed with 5 ,ul of plasmid DNA, 10 ,ul of 10 mg/ml sonicated and denatured salmon sperm carrier DNA, and 100 ,ul of 70% PEG in TE. After 30 min, the cells were heat shocked for 15 min at 42°C, diluted with 200 ,ul of TE, and directly plated onto selective medium.
Mapping of end6-1 The strain RH2063 carries a copy of ADP1 that is disrupted with URA3 and it also carries HIS4, trpl, MATa, and END6 (Table 1A). The trpl marker is centromere linked and thus is useful for scoring recombination between end6-1 and CEN3. This strain was mated with RH2082 (ADP1 ura3 his4 end6-1 TRP1 MATa) to create the multiply heterozygous diploid RH2071 (Table 1A). This diploid was sporulated and 98 tetrads were analyzed. Unfortunately, it was not possible to score the Ts- phenotype associated with end6-1 in many of the end6-1 spores in this cross, presumably because the RH2063 strain carries a suppressor of this phenotype. Analysis of this cross, however, allowed us to map adpl::URA3 accurately with respect to CEN3, MAT, and HIS4. We could score the Ts- phenotype conferred by the end6-1 mutation in all end6-1 spores obtained during outcrossing of the end6-1 mutation, where trpl, end6-1, and MAT were segregating in the RH144-3D background (Table 1A). Analysis of 24 tetrads allowed us to place end6-1 approximately 23 cM from MAT, and approximately
