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Vol. 20, Issue 5, 1312-1323, March 1, 2009
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Life Sciences Institute, Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2216
Submitted September 19, 2008;
Revised December 14, 2008;
Accepted December 22, 2008
Monitoring Editor: Janet M. Shaw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cells. These findings suggest that PTC1 promotes the association of myosin-V with its organelle-specific adaptor proteins. Moreover, these observations suggest that despite the existence of organelle-specific receptors, there is a higher order regulation that coordinates the movement of diverse cellular components. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Early in the cell cycle, a portion of each organelle is transported into the emerging bud. The polarized transport of most organelles from the mother to the bud is an active process that requires the actin cytoskeleton, myosin-V motors, and receptor proteins, which physically connect the motor to organelle cargoes (Beach et al., 2000; Yin et al., 2000; Boldogh et al., 2001; Barr, 2002; Bretscher, 2003; Du et al., 2004; Fagarasanu et al., 2006b, 2007; Weisman, 2006). Thus, formation of a complex between the motor and receptor protein is important for polarized organelle transport.
Similar to yeast, in vertebrates, myosin-V motors move cargoes along the actin cytoskeleton. The best studied cargo of vertebrate myosin-V are melanosomes, which are moved in melanocytes by myosin-Va. Melanosomes attach to myosin-Va through Rab27a and melanophilin (Fukuda and Kuroda, 2002; Wu et al., 2002). Rab27a, through geranylgeranylation, attaches to the melanosome membrane, and melanophilin connects myosin-Va and Rab27a. Myosin-V–based intracellular movement has been analyzed in many other eukaryotes, including frogs, fish, mammals, and plants; plant myosin-XI is the functional homologue of yeast and vertebrate myosin-V (Kinkema and Schiefelbein, 1994; Provance and Mercer, 1999; Tuma and Gelfand, 1999; Desnos et al., 2007; Li and Nebenfuhr, 2007; Sheets et al., 2007; Shimmen, 2007).
Genetic screens have identified several molecules that play a role in organelle inheritance. For example, vacuole inheritance utilizes the actin cytoskeleton and the class-V myosin complex, composed of Myo2p, Vac17p, and Vac8p (Hill et al., 1996; Wang et al., 1998; Ishikawa et al., 2003; Tang et al., 2003). Myo2p is one of two class-V myosin motors in budding yeast. Vac17p links Myo2p to the vacuole. Regulation of VAC17 likely plays a critical role in the control of vacuole movement. Protein and mRNA levels of VAC17 are tightly regulated during the cell cycle (Spellman et al., 1998; Zhu et al., 2000; Tang et al., 2003). In addition, phosphorylation of Vac17p plays a role in the initiation of vacuole inheritance (Peng and Weisman, 2008). Increased synthesis of VAC17 is likely a key event in the initiation of vacuole movement. Likewise, degradation of Vac17p is required for the termination of vacuole inheritance and the detachment of Myo2p from the vacuole membrane (Tang et al., 2003). Vac8p binds directly to Vac17p and attaches to the vacuole membrane via myristoylation and palmitoylation (Wang et al., 1998; Peng et al., 2006; Tang et al., 2006). Although the regulation of VAC17 contributes to the control of vacuole inheritance, other members of the myosin-V transport complex are also potential targets for the regulation of vacuole movement.
Other yeast organelles are also moved by myosin-V. Mitochondria are moved in part by Myo2p through interaction with Mmr1p and Ypt11p (Itoh et al., 2002, 2004; Boldogh et al., 2004; Altmann et al., 2008; Valiathan and Weisman, 2008). Peroxisomes are moved by a complex of Myo2p and the peroxisomal membrane protein, Inp2p (Fagarasanu et al., 2006a). Late-Golgi elements are moved by Myo2p with Ypt11p, a Rab GTPase, and Ret2p, a subunit of the coatomer complex (Rossanese et al., 2001; Arai et al., 2008). Secretory vesicles are moved by Myo2p (Pruyne et al., 1998; Schott et al., 1999) and require the Rab GTPases Ypt31p and Ypt32p (Casavola et al., 2008; Lipatova et al., 2008). Spindle orientation is also regulated by Myo2p and requires Kar9p, which connects Myo2p to Bim1p and subsequently to the ends of cytoplasmic microtubules (Beach et al., 2000; Yin et al., 2000). Similarly, the cortical ER is moved by Myo4p, the other yeast myosin-V, which forms a complex with She3p (Estrada et al., 2003). Several mRNAs are also moved by Myo4p and require both She3p and She2p, proteins whose roles are not well established (Bohl et al., 2000; Long et al., 2000; Takizawa et al., 2000; Takizawa and Vale, 2000; Shepard et al., 2003; Heuck et al., 2007; Hodges et al., 2008). Moreover, rather than attachment to the globular tail, She3p binds the rod of Myo4p. Thus, the mechanism of cargo attachment for Myo4p is likely to be distinct from Myo2p.
Mutations that completely block vacuole inheritance, myo2-2, vac8, and vac17, led to the discovery of the core machinery that links the vacuole to the actin cytoskeleton. However, little is known about the genes that control vacuole movement. One candidate for the control of vacuole movement is PTC1. PTC1, a gene encoding a type 2C serine/threonine protein phosphatase, is required for the temporal control of the distribution of mitochondria, the cortical ER, and the vacuole (Roeder et al., 1998; Du et al., 2006). However the precise function of Ptc1p in these processes is unknown.
In addition to organelle inheritance, Ptc1p plays a role in the regulation of the high osmolarity glycerol (HOG) pathway (Martin et al., 2005). The HOG pathway is required for yeast to survive under hyperosmotic conditions and heat stress (Winkler et al., 2002; Westfall et al., 2004). Hog1p is regulated by both activation and inactivation through its phosphorylation and dephosphorylation, respectively. Activation of the HOG pathway is regulated by a mitogen-activated protein kinase (MAPK) cascade, which leads to the phosphorylation of the MAPK, Hog1p. Ptc1p, which is recruited to the MAPK kinase Pbs2p through interaction with Nbp2p, inactivates Hog1p by dephosphorylating it (Warmka et al., 2001; Mapes and Ota, 2004). Both Ptc1p and Nbp2p also regulate the cell wall integrity (CWI) MAPK pathway (Ohkuni et al., 2003b). PTC1 regulates cortical ER localization via the CWI pathway, but not the HOG pathway (Du et al., 2006). The CWI pathway is not involved in vacuole inheritance, and the HOG pathway is not involved in PTC1 regulation of vacuole or mitochondria distribution (Roeder et al., 1998; Du et al., 2006).
Here we report the isolation of a new mutant, vac10, which causes a defect in vacuole inheritance. The vac10 mutation is a missense mutation in the catalytic domain of PTC1. This mutation and several additional alleles demonstrate that Ptc1p phosphatase activity is essential for vacuole inheritance. Moreover, we show that in addition to regulating the distribution of mitochondria, the vacuole, and cortical ER, Ptc1p regulates the distribution of several additional cargoes of myosin-V motors including peroxisomes, secretory vesicles, and ASH1 mRNA. Consistent with Ptc1p regulation of multiple cargoes, in ptc1 cells both Myo2p and Myo4p are mis-localized. Surprisingly, loss of Ptc1p also affects several organelle-specific myosin-V adaptor proteins. These findings suggest that PTC1 regulates the assembly of myosin-V organelle-specific receptor complexes. In support of this hypothesis, we find that a fusion protein of Myo2-Vac17p rescues vacuole inheritance in a ptc1 mutant. These results suggest that PTC1 regulates intracellular movement and/or distribution of multiple cargoes, via regulation of the interaction of the molecular motors with the corresponding receptor proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Longtine et al., 1998; Nagai et al., 2002; Huh et al., 2003). Myo2p- and Myo4p-Venus strains were made using plasmid pBS7 (Nagai et al., 2002). Vac17p-3xGFP strain was made using plasmid PB1960 (from Dr. David Pellman, Harvard). Yeast cultures were grown at 24°C unless stated otherwise. Yeast extract-peptone-dextrose (YEPD; 1% yeast extract, 2% peptone, and 2% dextrose), synthetic complete (SC) lacking the appropriate supplement(s), and 5-FOA media were made as described (Kaiser et al., 1994). Unless stated otherwise, SC medium contained 2% dextrose. When indicated, SC medium without uracil was supplemented with 0.5% casamino acids (Difco, Becton Dickinson; Franklin Lakes, NJ).
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) to generate pRS416-PTC1 or pRS415-PTC1, respectively. For pRS416-GFP-PTC1, a BglII site was generated by site-directed mutagenesis using the following primers: 5'-CTT TTA AAA ATC ATT ATA ATG AGa tcT CAT TCT GAA ATC TTA GAA-3' and 5'-TTC TAA GAT TTC AGA ATG Aga tCT CAT TAT AAT GAT TTT TAA AAG-3' to generate pRS416-PTC1-BglII. Green fluorescent protein (GFP) was amplified by PCR from pFA6a-GFPS65S-KanMX6 (Longtine et al., 1998) as a template using the following primers: 5'-GGG AGA TCT ATG AGT AAA GGA GAA GAA CTT TTC-3' and 5'-CGC GGA TCC TTT GTA TAG TTC ATC CAT GC-3'. The GFP fragment was inserted into pRS416-PTC1-BglII at the BglII site to generate pRS416-GFP-PTC1. For pRS416-GFP-ptc1-D58N, -D272N, -E36A/D37A, and -D233A, the PTC1 gene in pRS416-GFP-PTC1 was mutagenized by site-directed mutagenesis using the following primers: D58N-S: 5'-GGA TAT TTC GCG GTG TTT aAT GGA CAT GCT GGG ATT CAG-3'; D58N-AS; 5'-CTG AAT CCC AGC ATG TCC ATt AAA CAC CGC GAA ATA TCC-3'; D272N-S: 5'-GCT TTG GAA AAT GGC ACA ACA aAT AAT GTA ACG GTC ATG GTT GTC-3'; D272N-AS: 5'-GAC AAC CAT GAC CGT TAC ATT ATt TGT TGT GCC ATT TTC CAA AGC-3'; E36A/D37A-S: 5'-CTC GAA ATT TCG GAG GAC AAT GGc AGc TGT TCA TAC GTA TG-3'; E36A/D37A-AS: 5'- CAT ACG TAT GAA CAc CTc CCA TTG TCC TCC GAA ATT TCG AG -3'; D233A-S: 5'- CAA ATT TTT AAT CCT AGC GTG TGc TGG ATT ATG GGA TGT TAT TG-3'; D233A-AS; and 5'-CAA TAA CAT CCC ATA ATC CAc CAC ACG CTA GGA TTA AAA ATT TG-3'. The mutated nucleotides are in lower case.
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). Second, full-length VAC17 missing the initiating methionine was amplified by PCR using primers 5'- CGA gct agc GCA ACC CAA GCC CTA GAG-3' and 5'-AGG tct aga TTA AAA CAG CAG TTC TGT ATT CAA AGC-3'. The VAC17 fragment was inserted into the pBS myo2-tail-NheI at the NheI site to generate pBS myo2-tail-VAC17. Finally, an EcoRI-EcoRI fragment was subcloned from pBS myo2-tail-VAC17 into pRS415 MYO2 EcoRI-EcoRI.
In Vivo Labeling of Vacuoles
Vacuoles were labeled in vivo with N-(3-triethelammoniumpropyl)-4-(6 (4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM4-64; Molecular Probes, Eugene, OR) essentially as described (Ishikawa et al., 2003). In brief, a 2 mg/ml stock solution of FM4-64 in DMSO was added to early log phase cultures for a final concentration of 80 µM. After 1 h of labeling, cells were washed and were then chased in fresh liquid medium for 3–4 h.
Fluorescence Microscopy
Images were obtained using the DeltaVision RT Restoration Microscopy System (Applied Precision, Issaquah, WA).
Western Blot Analysis
SDS-PAGE and Western blot analysis were performed using standard procedures. Primary and secondary antibodies were used at the following concentrations: affinity-purified goat anti-Myo2p-tail (1:3000), HRP-donkey anti-goat IgG (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA), affinity-purified sheep anti-Vac17p (1:3000), HRP-donkey anti-sheep IgG (1:5000; Sigma, St. Louis, MO), affinity-purified rabbit anti-Vac8p (1:5000), HRP-goat anti-rabbit IgG (1:5000; Jackson ImmunoResearch Laboratories), mouse anti-GFP (1:5000; Roche, Indianapolis, IN), HRP-goat anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories), mouse anti-Pgk1p (1:20,000; Invitrogen, Carlsbad, CA), and HRP-goat anti-mouse IgG (1:20,000; Jackson ImmunoResearch Laboratories). HRP activity was detected using ECL plus (Amersham Bioscience, Piscataway, NJ).
RNA Preparation and RT-PCR
Total RNA from yeast was prepared by the glass bead method as described (Mizuki et al., 2007). Cells were harvested and disrupted by mixing vigorously with glass beads in TELS solution containing 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 100 mM LiCl, and 1% SDS and an equal volume of phenol/chloroform/isoamylalcohol (PCI). After centrifugation, the lysate was treated with an equal volume of PCI. Ethanol was then added and the mixture was centrifuged at 12,000 rpm at 4°C for 20 min to precipitate the RNA. RNA was dissolved in water. cDNA was prepared by reverse transcription of total RNA using oligo dT primer (Bio-Rad, Hercules, CA). RT-PCR was performed using standard procedures using the following oligonucleotides: VAC17, 5'-GCC AGA CAA CAG ATC AAG AG-3' and 5'-TAG GTG AGC ACG GTA AAG AG-3'; and PGK1, 5'-CCA AGA TTT GGA CTT GAA GG-3' and 5'-AAA CAT CAG CCA AAG AGC TC-3'.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We identified a genomic plasmid with 9000 bases, spanning part of the genomic region of chromosome IV that contained seven genes including PTC1 (http://www.yeastgenome.org/). PTC1 was the only ORF in the genomic plasmid that suppressed the vacuole inheritance defect of the vac10-1 mutant (data not shown). In addition both the ptc1 and vac10-1 mutants have the same phenotype: defects in vacuole inheritance and fragmented vacuoles (Figure 1, A and B; Wang et al., 1996; Bonangelino et al., 2002; Du et al., 2006). We found a single missense mutation in vac10-1: ptc1-D272N (Supplemental Figure S1). A plasmid encoding ptc1-D272N did not suppress the vacuole inheritance (Figure 1C) or fragmented vacuole defect in vac10-1 (data not shown). These results indicate that the corresponding gene of vac10-1 is PTC1 and that PTC1 plays a role in vacuole inheritance.
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mutant is due to a defect in vacuole movement or in the retention of vacuoles in the bud, we performed time-lapse analysis of vacuole inheritance in wild-type or ptc1 cells. We collected 10 time-lapse images ranging from 24 to 120 min, for both wild-type and ptc1 cells, where we observed cells that initially contained no vacuole in the bud. For each time-lapse series of wild-type cells, we observed a vacuole moving from the mother to the bud. In contrast, nine of the 10 time-lapse sequences for ptc1 cells showed no vacuole movement to the daughter cell; one time-lapse sequence showed normal vacuole inheritance. In this one example the vacuole remained in the bud for the rest of the observation time, during which the diameter of the bud grew to half the diameter of the mother (data not shown). These results indicate that PTC1 is required for vacuole movement and is not likely required for retention of the vacuole in the bud (Figure 2 and Supplemental Figure S2).
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cells are unlikely due to defects in the actin cytoskeleton. Actin cables and patches are normal in ptc1 cells (Supplemental Figure S3; Roeder et al., 1998; Du et al., 2006).
The Phosphatase Activity of Ptc1p Is Required for Vacuole Inheritance
PTC1 encodes a type 2C protein phosphatase. There are seven type 2C protein phosphatases in the S. cerevisiae genome, PTC1- 7; each has protein phosphatase activity in vitro (Cheng et al., 1999; Jiang et al., 2002; Ruan et al., 2007). Type 2C protein phosphatases have a metal-binding site that is required for phosphatase activity (Supplemental Figure S1; Das et al., 1996). Notably the vac10-1 mutation D272N is in a conserved metal-binding residue, thus it is likely that vac10-1 does not have phosphatase activity.
To test further whether the phosphatase activity of Ptc1p is required for vacuole inheritance, we generated a GFP fusion protein of Ptc1p-D58N, a mutant that was previously shown to lack phosphatase activity in vitro (Warmka et al., 2001). We also generated GFP fusion proteins of vac10-1 (D272N) and two predicted phosphatase-dead mutants, GFP-Ptc1p-E35A/D36A and -D233A. The known phosphatase-dead mutant GFP-Ptc1p-D58N as well as the predicted phosphatase-dead mutants did not complement the vacuole inheritance defect of ptc1 (Figure 3A). Note that all GFP-Ptc1 fusion proteins had normal levels of expression (Figure 3B). These results strongly suggest that the phosphatase activity of Ptc1p is essential for vacuole inheritance.
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; this study), PTC1 regulates the distribution of mitochondria and the cortical ER (Roeder et al., 1998; Du et al., 2006). These organelles attach to either Myo2p or Myo4p. To test whether Ptc1p regulates the distribution of all known cargoes that attach to either Myo2p or Myo4p, we tested the distribution of the known cargoes of Myo2p, including secretory vesicles, peroxisomes, the late-Golgi and also mitotic spindle orientation. As a marker of secretory vesicles, we expressed GFP-Sec4p under its endogenous promoter. In wild-type cells, GFP-Sec4p localized to the bud tip or incipient bud site (Figure 4A; Calero et al., 2003). In contrast, in ptc1 cells, GFP-Sec4p was mis-localized (Figure 4A). This suggests that PTC1 is involved in secretory vesicle movement. In further support of this postulate, ptc1 cells have a slow-growth phenotype (Figure 4B; Roeder et al., 1998), a phenotype that is also observed in other secretion mutants (Novick et al., 1980). To test peroxisome inheritance, we expressed GFP fused to peroxisomal targeting signal 1 (GFP-PTS1) in wild-type, myo2-66, or ptc1 cells. In wild-type cells, almost every bud with a diameter less than half the diameter of the mother contained peroxisomes (Figure 4C). In contrast, similar to myo2-66, ptc1 cells show a defect in peroxisome distribution; only 55% of small buds showed normal peroxisome distribution (Figure 4C). To determine whether there was a delay or defect in peroxisome inheritance, we assigned cells to one of three classes based on bud size: small, medium, and large. All classes showed a defect (Supplemental Figure 4). Thus PTC1 is involved in the distribution of some of the organelles moved by Myo2p, including the vacuole, mitochondria, peroxisomes, and secretory vesicles.
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cells. Wild-type and ptc1 cells showed normal late-Golgi distribution (Figure 5A). To test mitotic spindle orientation, we expressed GFP fused to tubulin (GFP-Tub1p) under its endogenous promoter in wild-type, ptc1, or kar9 cells. Wild-type and ptc1 cells showed normal mitotic spindle orientation (Figure 5B). The fact that Myo2p movement of some cargoes is not affected indicates that actin–myosin interactions are normal in the ptc1 mutant.
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cells (Du et al., 2006). We tested whether Ptc1p regulates the other known cargoes of Myo4p, such as specific mRNAs and used the best-characterized example, ASH1 mRNA. We coexpressed MS2, the bacteriophage MS2 coat RNA-binding protein as a GFP fusion protein and RNA that has the ASH1 3' UTR with MS2-binding sites (Beach et al., 1999). The ASH1 reporter RNA can be visualized with the MS2-GFP signal. In wild-type cells, 26% of cells with small buds have a localized GFP signal at the bud tip (Figure 5C; Beach et al., 1999). In contrast, in ptc1 cells, only 17% of the small-budded cells have a localized GFP signal at the bud tip (Figure 5C). Thus, PTC1 also regulates the localization of the Myo4p cargo, ASH1 mRNA.
PTC1 Affects Both Myosin-V Motors and Organelle-specific Receptors
That most of the cargoes moved by yeast myosin-V motors require Ptc1p suggested the possibility that both Myo4p and Myo2p are regulated by Ptc1p. Therefore we tested the localization and expression levels of Myo4p and Myo2p. We constructed strains with genes that encode Myo4p-Venus or Myo2p-Venus integrated into the correct chromosomal locus. In wild-type cells, Myo4p is localized to the bud tip (Figure 6A; Jansen et al., 1996; Wesche et al., 2003). In contrast, Myo4p-Venus was mis-localized in the ptc1 mutant and was diffusely spread throughout the bud (Figure 6A). Myo2p-Venus was also partially mis-localized in ptc1 cells (Figure 6B); there was some loss of concentration of Myo2p at the bud tip. The protein expression levels of Myo4p-Venus and Myo2p-Venus were normal (Figure 6, C and D).
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cells (Figure 7, A and B). These results suggest that PTC1 is required the normal steady-state levels of several organelle-specific receptors.
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; Hodges et al., 2008). Thus, Myo4p attaches to cargoes via a mechanism that is distinct from Myo2p and currently is not well established. Therefore we focused on the role of Ptc1p in the attachment of Myo2p to its known cargoes.
PTC1 Is Required for the Proper Association of the Vacuole Transport Complex
Similar to Myo2p mis-localization in ptc1 cells, Myo2p is also mis-localized in myo2 mutants that are defective in binding cargoes. Both myo2–2p, which is defective in binding Vac17p and myo2p-Y1415E, which is defective in binding Ypt31/32p, are mis-localized (Catlett et al., 2000; Lipatova et al., 2008; data not shown). These observations suggest the possibility that PTC1 regulates organelle inheritance by controlling the association of the Myo2p with organelle-specific receptors. Because Myo2p association with the vacuole-specific transport complex is the best characterized, we chose to further investigate the impact of Ptc1p on the association of Myo2p with Vac17p and Vac8p.
As a first approach, to determine why Vac17p levels are reduced in ptc1 cells, we tested the steady-state levels of VAC17 mRNA and Vac17p protein. Using semiquantitative RT-PCR, we found that the levels of VAC17 mRNA are the same in ptc1 and wild-type cells (Figure 7C). This result is consistent with a transcriptome-based analysis of the roles of type 2C protein phosphatases in budding yeast (Gonzalez et al., 2006). The steady-state levels of Vac17p were also reduced in the phosphatase-dead ptc1-D58N mutant (Figures 3B). These results suggest that through its phosphatase activity, Ptc1p is required to maintain the steady-state levels of Vac17p.
Ptc1p functions with Nbp2p in several signaling pathways (Ohkuni et al., 2003b; Mapes and Ota, 2004; Du et al., 2006), and Nbp2p also regulates the distribution of the vacuole and cortical ER (Du et al., 2006). The ptc1 and nbp2 strains share the same phenotypes including a defect in vacuole inheritance and fragmented vacuoles (Du et al., 2006; Supplemental Figure S7). We tested and found that the steady-state levels of Vac17p were also reduced in nbp2 cells (Figure 7D).
To determine whether the defect in vacuole inheritance in ptc1 cells is due to the reduction of Vac17p, we overexpressed VAC17 in the ptc1 mutant. We found that overexpression in ptc1 cells of Vac17p, but not Vac8p, partially rescued the vacuole inheritance defect (Figure 7, E–G, and Supplemental Figure S5). Therefore, the decrease in Vac17p contributes to the vacuole inheritance defect in ptc1. Note that the fragmented vacuole phenotype of the ptc1 mutant was not restored by overexpression of Vac17p (Figure 7E). Thus the role of Ptc1p in vacuole inheritance is likely distinct from its role in vacuole fusion.
To test whether Ptc1p regulates the localization of Vac17p, we constructed a yeast strain that encodes Vac17p-3xGFP expressed from the correct chromosomal locus. Vac17p-1xGFP could not be detected because on average only 20 Vac17p molecules are present per cell (Tang et al., 2006). In wild-type cells, Vac17p-3xGFP is localized to the vacuole membrane and is concentrated at the leading edge of the vacuole (Figure 8A). In the ptc1 mutant, although Vac17p-3xGFP was localized on the vacuole membrane, it was distributed throughout the vacuole membrane (Figure 8A). Vac17p is similarly mis-localized in the myo2-N1304S mutant, which is defective in binding to Vac17p. Together, these results strongly suggest that PTC1 is required for the proper association of Myo2p and Vac17p. Note that the localization of Vac8p was unaffected in ptc1 cells (Supplemental Figure S5).
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; Legesse-Miller et al., 2006), and more recently we found that Vac17p is also phosphorylated (Peng and Weisman, 2008). We tested the phosphorylation status of all three proteins. Phosphorylation of Myo2p and Vac8p is clearly unchanged (Supplemental Figure S6). Notably there was less phosphorylated Vac17p. However if Ptc1p acts directly on Vac17p, we would predict that loss of a phosphatase would lead to higher phosphorylation. One potential problem with analyzing the phosphorylation of Vac17p is that there is also significantly less Vac17p, perhaps because of its instability. Thus, it is possible that in a ptc1 strain a phosphorylated form of Vac17p is not dephosphorylated but is also not detected because it is rapidly turned over. Thus, we cannot conclude that Vac17p is not a direct target of Ptc1p.
If PTC1 regulates the association between Myo2p and Vac17p, strengthening the association between Myo2p and Vac17p would rescue the defect of vacuole inheritance in ptc1 cells. To test this hypothesis, we expressed Vac17p fused to the cargo-binding domain of Myo2p in the ptc1 mutant. The fusion protein, Myo2-Vac17p partially rescued the vacuole inheritance defect in ptc1 cells (Figure 8B). Note that the steady-state levels of the Myo2-Vac17 fusion protein were significantly lower than Vac17p (Figure 8C). Thus, the suppression by Myo2-Vac17p was not due to overexpression of Vac17p. Moreover, Vac17p fused to the cargo-binding domain of myo2p-N1304S mutant, which has defect of binding to Vac17p, also rescued the defect in the ptc1 mutant (Figure 8B). This indicates that the suppression by the Myo2-Vac17 fusion protein is not due to an additional Vac17 protein binding to Myo2p. These results strongly suggest that PTC1 controls the association of the vacuole transport complex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ptc1p is the only serine/threonine phosphatase known to affect organelle transport in yeast (Roeder et al., 1998; Du et al., 2006; this study). Previous studies showed that PTC1 is required for the distribution of the mitochondria and the vacuole, cargoes moved by Myo2p, and for distribution of the cortical ER, a cargo moved by Myo4p. That the phosphatase activity of Ptc1p is required for proper distribution of the cortical ER (Du et al., 2006) and the vacuole (Figure 3) strongly suggests that Ptc1p phosphatase activity per se is required for the regulation of each organelle. Thus, dephosphorylation by Ptc1p regulates the activities of one or more proteins required to move intracellular cargoes.
To gain insight into the mechanism of Ptc1p regulation, we focused specifically on Myo2p and the vacuole-specific receptor protein, Vac17p. We tested and found that loss of Ptc1p results in a defect in the association of Myo2p and Vac17p. The proportion of Vac17p that is bound to Myo2p is reduced in ptc1 cells (Supplemental Figure S8). Moreover, a fusion protein of Myo2-Vac17p rescued the defect of vacuole inheritance in ptc1 cells (Figure 8B). The observation that both Myo2p and Vac17p are mislocalized is consistent with a defect in the association of the Myo2p-Vac17p complex. When myosin motors are defective in forming organelle-specific complexes, the motor and organelle specific receptor are mis-localized (Catlett et al., 2000; Lipatova et al., 2008; Peng and Weisman, 2008).
Surprisingly, the levels of Vac17p in the ptc1 mutant are significantly lower than that in a wild-type strain (Figure 7B). This is the opposite of what occurs in other mutants that disrupt the Myo2p-Vac17p complex. For example, in the myo2-N1304S mutant, Vac17p levels are elevated. Because we were unable to determine the direct down stream targets of Ptc1p, we could not determine how Vac17p is destabilized. It is tempting to speculate that Ptc1p functions to promote the stabilization of Vac17p. Alternatively, in the ptc1 strain, the phosphorylation status of Vac17p may be altered in a way that makes it a target for proteolysis, either through its normal pathway or through other pathways. Another possibility is that loss of Ptc1p may result in less Vac17p, which then leads to less of the Myo2p-Vac17p complex.
We sought to narrow down the above alternatives by testing the levels of Vac17p in a ptc1/myo2-N1304S double mutant. However we found that a combination of these two mutations is synthetically lethal (Supplemental Table S1). Moreover, myo2–2, another point mutation that disrupts Myo2p-Vac17p interactions, is also synthetically lethal with ptc1. The synthetic lethality appears to be specific to mutations in Myo2p that reside within the Vac17p-binding site. We found that ptc1 is not synthetically lethal with myo2-66 (Supplemental Table S1), which is due to point mutation in the actin-binding domain.
PTC1 Is Involved in Several Signaling Pathways with NBP2
PTC1 is a negative regulator of both the HOG and the CWI MAP kinase signaling pathways (Huang and Symington, 1995; Warmka et al., 2001). In both pathways, Ptc1p functions with Nbp2p, a direct binding partner of Ptc1p.
If HOG1 plays a role in Ptc1p regulation of vacuole inheritance, then deletion of Hog1p should restore the loss of vacuole inheritance caused by loss of Ptc1p. However, the ptc1/hog1 double mutant did not restore vacuole inheritance, and the single hog1 mutant had normal vacuole inheritance (Du et al., 2006; data not shown). Thus, Ptc1p regulation of vacuole inheritance is unlikely to be mediated through the HOG pathway. Moreover, the HOG pathway is not involved in either mitochondria inheritance or cortical ER inheritance (Roeder et al., 1998; Du et al., 2006).
PTC1 is also a negative regulator of the MAP kinase SLT2/MPK1, a component of the CWI pathway (Huang and Symington, 1995; Ohkuni et al., 2003a; Gonzalez et al., 2006). If SLT2 were involved in vacuole inheritance, SLT2 would act as a negative regulator and the double mutant, ptc1/slt2, should have normal vacuole inheritance.
Unfortunately, in our strain background, the ptc1/slt2 double mutant has a severe growth defect; thus vacuole inheritance could not be assessed. However, an earlier study showed that vacuole inheritance in a ptc1/slt2 double mutant was the same as in a ptc1 mutant (Du et al., 2006). In addition, we found that Vac17p levels are decreased in both ptc1/hog1 and ptc1/slt2 double mutants (data not shown). These results suggest that neither the HOG nor CWI pathways regulate vacuole inheritance and that PTC1-NBP2 regulates vacuole inheritance, and perhaps the movement of other Myo2p cargoes, through an as yet undetermined pathway (Model Figure 9). In contrast, PTC1 and NBP2 regulate cortical ER inheritance via the CWI pathway (Du et al., 2006).
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Biology, University of Arkansas, Little Rock, AR 72204-1099. 
Address correspondence to: Lois S. Weisman (lweisman{at}umich.edu)
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, normal vacuole inheritance; 
, bud without detectable FM4-64; 
, weaker FM4-64 signal in the bud than in the mother cell. (C) The vac10-1 mutant is due to a missense mutation in PTC1. The ptc1-D272N mutant does not complement vac10-1. (B and C) Error bars, SDs calculated from three experiments.
90), medium- (
