Article |
Address correspondence to Lois S. Weisman, Dept. of Biochemistry, The University of Iowa, Iowa City, IA 52242. Tel.: (319) 335-8581. Fax: (319) 335-9570. E-mail: lois-weisman{at}uiowa.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: membrane transport; Myo2p; Vac17p; yeast; vacuole
N.L. Catlett's present address is Torrey Mesa Research Institute, Syngenta Research and Technology, 3115 Merryfield Row #100, San Diego, CA 92121.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The globular tail of myosin V plays an important role in its attachment to cargo. Overexpression of the globular tail of myosin Va causes a defect in melanosome movement (Wu et al., 2002a); likewise, overexpression of the globular tail of Myo2p disrupts secretory vesicle targeting and causes cell death (Reck-Peterson et al., 1999; Schott et al., 1999). In both cases, overexpression of this domain is thought to compete with endogenous myosin V and displace it from cargo. Additionally, cell cyclespecific phosphorylation of the myosin Va globular tail releases it from membranes (Karcher et al., 2001), suggesting that post-translational modification of the tail may be important for its attachment to cargo.
The globular tail of Myo2p contains at least two distinct cargo-binding domains, one specific for vacuole movement (Catlett and Weisman, 1998; Catlett et al., 2000), the other specific for secretory vesicles (Schott et al., 1999; Catlett et al., 2000). The vacuole-specific region was defined by seven point mutations affecting one of five amino acids between residues 12481307 of the Myo2p globular tail domain. The secretory vesicle binding domain was identified via sequence analysis of a set of myo2ts mutants (Schott et al., 1999), and by identification of myo2-14591491, a mutant specifically defective in secretory vesicle movement (Catlett, 2000). Overexpression of the Myo2p globular tail missing the secretory vesiclespecific region disrupts vacuole inheritance, but does not affect secretory vesicle targeting. Conversely, mutations in the vacuole-binding domain cause defects in vacuole movement, but do not affect other Myo2p-related functions such as secretion. Given that specific regions of the globular tail are required for different functions of Myo2p, the existence of cargo-specific receptors for Myo2p was predicted. Here, we describe Vac17p, a novel protein that is a key component of the vacuole-specific Myo2p receptor.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surprisingly, VAC17-S57F suppressed the vacuole inheritance defect of all the vacuole-specific myo2 tail mutants (Fig. 1 and Table I). These findings prompted us to perform a directed screen for other mutations in VAC17 that could suppress the vacuole inheritance defect of myo2-N1304S. We identified a second amino acid substitution (VAC17-I140V) that showed allele specificity, as it only suppressed the vacuole inheritance defects of myo22, myo2-N1304S, and myo2-N1307D (Fig. 1 and Table I).
|
|
|
|
Deletion analysis of Myo2p showed that removal of residues 12971307 abolished Vac17pMyo2p interactions (Fig. 3 C). However, this short sequence is not sufficient for Vac17p binding, as the shortest segment of the tail that interacts with Vac17p (residues 11131568) encompasses nearly the entire globular tail (Fig. 3 C).
To determine if the vacuole inheritance defects seen in the myo2 tail mutants result from poor interaction of the mutant Myo2p with Vac17p, we examined interaction of Vac17p (1355) with myo2p tail fusions of five representative mutants (myo22/G1248D, D1297N, L1301P, N1304D, and N1304S). None of these constructs interacts significantly with Vac17p (Fig. 3 D). Western blot analysis indicates that all of these mutant Myo2p fusion proteins were expressed to a similar degree as wild type (Fig. 3 E). Together with the requirement of Vac17p in vacuole inheritance, these observations suggest that the interaction of Vac17p and Myo2p is normally required for vacuole movement.
We further analyzed the relationship between Vac17pMyo2p interactions and vacuole inheritance by testing the interaction of additional myo2 mutants with Vac17p. As part of a separate paper (Catlett, N.L., unpublished data), we obtained a collection of intragenic suppressors of the myo22 (G1248D) vacuole inheritance defect via random PCR mutagenesis. Eight of these suppressors restore vacuole inheritance to >80% when present as the sole copy of MYO2. Each suppressor, except the pseudorevertant G1248N, contains both G1248D and a second point mutation. We constructed myo2 tail DNA binding-domain fusions of these suppressors and found that all eight simultaneously restored vacuole inheritance and Vac17pMyo2p interactions. This finding demonstrates that the vacuole inheritance defect in myo22 is directly related to a loss of Vac17pMyo2p interactions.
Analysis of VAC17-I140V also strongly supports the hypothesis that Vac17pMyo2p interactions are required for vacuole inheritance. The I140V mutation is located in the Myo2p binding site. VAC17-I140V restored vacuole inheritance in myo2-N1304S from 10 to 50% (Table I). Moreover, it showed allele specificity, restoring vacuole inheritance in myo22 (G1248D), N1304S, and N1307D, but not in other vacuole-specific myo2 point mutants (Table I). Next, we tested whether VAC17-I140V, in addition to restoring vacuole inheritance, restored interaction with myo22 and myo2-N1304S (Fig. 4 D). A VAC17-I140V two-hybrid construct (97260) restored interaction with both myo22 and myo2-N1304S. A longer fusion, VAC17-I140V (1260) also interacted with the globular tail domain of myo22. The suppression of myo22 by I140V is not due to overexpression of Vac17p because its levels are similar to the wild-type protein (unpublished data). Thus, VAC17-I140V suppresses selected vacuole-specific myo2 mutants by increasing the affinity of Vac17p for Myo2p through the original interaction site.
|
Although Vac17p interacts with Myo2p and also Vac8p (Tang et al., 2003), our analysis of VAC17-S57F suggested that Vac17p also interacts with yet another molecule to regulate Myo2p attachment to the vacuole. The molecule predicted by the behavior of VAC17-S57F is unlikely to be Vac8p because the Vac17p-S57F mutation maps outside of the Vac8p binding region (Tang et al., 2003). Moreover, VAC17 and VAC17-S57F interact with VAC8 to the same extent (unpublished data).
Evidence that Vac17p interacts with at least one additional molecule includes the following: First, VAC17-S57F showed no allele specificity, suppressing all of the vacuole-specific myo2 point mutants to a similar degree (Fig. 2 B). Second, although VAC17-S57F interacted with wild-type Myo2p, it did not restore interactions with the vacuole-specific Myo2p point mutants (Fig. 4 D). Furthermore, the S57F mutation maps outside of the Myo2p binding region of Vac17p (Fig. 4 C). Finally, this region of Vac17p is not essential for vacuole inheritance; removal of residues 197 from Vac17p reduces vacuole inheritance by only 30% (unpublished data). The most likely explanation for these data is that Vac17p-S57F indirectly restores Vac17pMyo2p interactions, suggesting that at least one other protein is involved in Myo2p binding to Vac17p. This protein could be part of the receptor complex and could work with Vac17p to promote Myo2p interaction with vacuole. Alternatively, the protein might negatively regulate Vac17pMyo2p interactions, but not Vac17p-S57FMyo2p interactions. The suppression by VAC17-S57F is not due to elevated Vac17p levels; the cellular concentration of Vac17p-S57F is the same as the wild-type protein (unpublished data). Also, it is unlikely that the S57F mutation now allows Vac17p to interact with Myo2p at a site distinct from the globular tail. Using the yeast two-hybrid test, we found no interaction between Vac17p-S57F and the Myo2p tail containing both the globular domain and the coiled-coil region or the coiled-coil region alone (unpublished data).
By definition, the vacuole-specific receptor for Myo2p would recruit the motor to the vacuole. Thus, using double-labeled immunofluorescence microscopy, we tested whether Vac17p is required for the association of Myo2p with vacuoles. In addition, we used Western analysis to measure the level of Myo2p on isolated vacuoles. For these experiments, WT, vac17, and VAC17-
PEST cells were compared. The removal of the PEST sequence stabilizes Vac17p, increasing the levels of Vac17p on the vacuole (Tang et al., 2003). This mutant, expressed from a low copy plasmid, was used instead of high copy VAC17 because the number of multicopy plasmids present varies widely from cell to cell. Like overexpression of Vac17p from a multicopy plasmid, VAC17-
PEST increases vacuole inheritance in myo22 from 11 to 37% (n = 187), but does not affect vacuole inheritance in myo2-N1304S (n = 232).
In wild-type cells, Myo2p concentrates at sites of polarized growth and is also present as small cytoplasmic spots, with a subset of spots colocalizing with the vacuolar membrane (Hill et al., 1996 and Fig. 5 A). Thus, the low levels of vacuolar Myo2p seen by immunofluorescence microscopy are insufficient to determine whether less Myo2p is present on vacuoles in vac17 cells. However, increasing Vac17p levels with the Vac17p-stabilizing mutant VAC17-
PEST (Tang et al., 2003) dramatically increased the levels of Myo2p on the vacuole (Fig. 5 C). Consistent with the immunofluorescence microscopy images, when compared with wild type, significantly higher levels of Myo2p copurified with VAC17-
PEST mutant vacuoles. Moreover, levels of Myo2p on vac17
vacuoles were significantly lower (Fig. 5 E). The above data strongly suggests that Vac17p is required for normal Myo2p association with the vacuole.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The molecular basis of Myo2p attachment to the vacuole shares similarities to what is currently known about myosin Va attachment to melanosomes. Melanophilin, a recently discovered rab effector, is a component of the melanosome-specific myosin Va receptor (Fukuda and Kuroda, 2002; Hume et al., 2002; Nagashima et al., 2002; Provance et al., 2002; Wu et al., 2002b). Melanophilin binds directly to myosin V, and interacts with melanosomes via interaction with Rab27a. Thus, both Vac17p and melanophilin bind directly to myosin V and attach to their respective membranes via interaction with an acylated protein.
Despite these similarities, there are several differences between attachment of myosin Va to melanosomes and Myo2p attachment to vacuoles. Melanophilin and Vac17p do not share any sequence similarity. Moreover, no obvious melanophilin homologues were found in the yeast genome database. Similarly, no Vac17p homologues were found in higher eukaryotes. In addition, Rab27a and Vac8p are not related proteins. Vac8p is not a small GTPase; rather, it likely plays its role via interaction with binding partners. It appears that Vac8p brings specific protein complexes to distinct regions of the vacuole (Wang et al., 2001). Moreover, although both Vac8p and Rab27a are acylated, Rab27a is geranylgeranylated at its COOH terminus, and Vac8p is myristylated and multiply-palmitoylated at is NH2 terminus. These diverse types of modifications are likely to serve distinct functions (Melkonian et al., 1999; Zacharias et al., 2002).
It is possible that the differences in these receptors arise because their respective membranes are found in distant organisms or because the melanosome is a specialized lysosomal-like organelle, whereas the yeast vacuole/lysosome serves more generalized functions.
Alternatively, these myosin V receptors may each contain additional proteins. A portion of the vacuole-specific region of Myo2p (residues 12911313) is weakly conserved with all vertebrate myosins and is highly conserved among the three classes of vertebrate myosin Vs (Catlett et al., 2000; unpublished data). This suggests that this region in vertebrate myosin Vs may bind a protein receptor. Melanophilin, which binds to the melanocyte-specific exon F that is outside of the globular tail domain, may also bind to this conserved region; alternatively there may be yet another protein that binds this region and functions together with melanophilin and Rab27a. Notably, both the globular tail domain plus exon F are needed to observe the dominant-negative effects caused by overproduction of the myosin Va tail (Wu et al., 2002b).
Similarly, the vacuole-specific receptor reported here may also include a specific rab protein. Rab27a has been shown to be required for myosin Va binding to melanosomes, and Rab11a is required for myosin Vb binding to recycling endosomes (Lapierre et al., 2001). Moreover, the rabs, Sec4p (Schott et al., 1999; Ortiz et al., 2002), and Ypt31/Ypt32 (Ortiz et al., 2002) may play a role in Myo2p movement of secretory vesicles.
The discovery of organelle-specific myosin V receptors demonstrates that myosin V attaches to membranes via proteinprotein interactions. Moreover, that Vac17p is not required for movement of other Myo2p cargo shows that within a single cell type, there are specific receptors for distinct membrane cargo. Perhaps these organelle-specific receptors compete with each other for access to the myosin V tail. Further study of Vac17pMyo2p interactions will help elucidate how organelle-specific receptors regulate myosin V attachment to its diverse cargoes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Screen for multicopy plasmids suppressing the myo22 vacuole inheritance defect
Multicopy library pools RB378 and RB380, derived from YEp24 (URA3, 2µ), and the pRS202-based library (URA3, 2µ) were gifts from Drs. David Botstein (Stanford University, Stanford, CA) and Philip Hieter (University of British Columbia, Vancouver, BC), respectively. pNLC16 (pGAL-PEP4-HIS3) was generated by subcloning the EcoRI/SalI fragment from a URA-based pGAL-PEP4 plasmid (Vida et al., 1990) into pRS413 (HIS3, CEN). LWY5518 yeast were cotransformed with pNLC16 and library DNA. Approximately 13,000, 45,000, and 19,000 transformants were screened from the pRS202 library and the RB378 and RB380 pools, respectively. Vacuole inheritance was assessed with the CPY plate assay. As expected, PEP4 was obtained; we also obtained MYO2. In addition, three isolates were obtained that restored vacuole inheritance to 30% when assessed by fluorescence microscopy. Each contained a plasmid with multiple ORFs including full-length VAC17 (YCL063W). VAC17 was identified as the corresponding wild-type gene for the vac mutant vac171, and VAC17 is required for vacuole inheritance (Tang et al., 2003). Thus, a multicopy plasmid containing VAC17 alone was tested, and suppressed the vacuolar inheritance of myo22 to a similar degree as the three candidate plasmids.
Screen for extragenic suppressors of the myo2-N1304S vacuole inheritance defect
myo2::TRP1 yeast carrying plasmids pmyo2-N1304S and pGAL-PEP4-URA3 were mutagenized with ethyl methanesulfonate as described previously (Winston, 1990). Vacuole inheritance was assessed with the CPY plate assay. VAC+ suppressor candidates were isolated, and the original pmyo2-N1304S plasmid was replaced with unmutagenized pmyo2-N1304S to distinguish between suppression due to a new mutation in the original myo2-N1304S plasmid or a mutation in another gene. One extragenic suppressor was identified out of 110,000 colonies. A heterozygous diploid of the suppressor candidate strain, with myo2-N1304S as the sole copy of MYO2, exhibited the same correction of vacuole inheritance as the haploid candidate strain, demonstrating that the suppressor mutation was dominant. In tetrads derived from this diploid, the restoration of vacuole inheritance segregated 2:2, indicating that the suppression arose from a single mutation. Therefore, a genomic library of this suppressor was constructed (see following paragraph) and transformed into the myo2-N1304S starting strain. Complementing plasmids were recovered and sequenced. The suppressing plasmid encoded VAC17 with a single point mutation (C170T), resulting in the amino acid substitution S57F.
Construction of the genomic suppressor library
myo2-N1304S was integrated into the genome of the suppressor strain (myo2::TRP1, pMYO2-URA3) via transformation and homologous recombination of a linear 5-kb fragment containing full-length myo2-N1304S (obtained from pmyo2-N1304S cut with ClaI, DraIII, and ScaI). The integrant was selected by growth on 5-fluoro-orotic acid plates and inability to grow on SC-TRP-URA plates. Size-fractionated (812 kb) genomic DNA from a partial Sau3A digest (techniques described in Nau et al., 1997) was ligated into the BamHI site of pRS415. Approximately 85% of the clones contained inserts.
Yeast two-hybrid analysis
Fusion of the GAL4 activation domain with VAC17 (pGAD-VAC17) is described in Tang et al. (2003). For pRS416-VAC17(1170), pRS416-VAC17 was cut with BstBI and religated, creating K171N, T172L, and N173-STOP. For pGAD-VAC17(1170), an 1.2-kb BglII and PacI fragment from pGAD-VAC17 was replaced with the corresponding fragment from pRS416-VAC17(1170). For pGAD-VAC17(97170), the
500-bp EcoRI fragment from pRS416-VAC17(1170) was cloned into the EcoRI site of pGAD-C1 (James et al., 1996). For pRS416-VAC17(1355), pRS416-VAC17 was cut with AflII, the sticky ends were filled in with Klenow, and the plasmid was religated, creating K356-STOP. For pGAD-VAC17(1355), the BglII and PacI fragment from pGAD-VAC17 was replaced with the corresponding fragment from pRS416-VAC17(1355). For the pGAD-VAC17(97260) plasmids, DNA was amplified from either pRS416-VAC17, pRS416-vac17-S57F, or pRS415-vac17-I140V using primers TF1v (5'-AAAAGGATCCATGGCAACCCAAGCCCTAGAG-3') and VAC17 Cla3R (5'-GGATCGATTTCAGCACCCTTTGCGGGCACACC-3'), which add BamHI and ClaI sites, respectively, and was ligated into pGAD-C1.
For the pGBD-MYO2(11131574) clones, DNA for all mutants and wild-type were PCR amplified from the relevant MYO2 plasmids (Catlett et al., 2000) using the MYO2-Bam3F (Catlett et al., 2000) and T3 universal primers. PCR products were cut with BamHI and ClaI and ligated into pGBD-C1 (James et al., 1996). To generate pGBD-myo2 11131574 14591491, the
1.5-kb BamHI and ClaI fragment of pNLC27 (Catlett, 2000) was subcloned into pGBD-C1.
The following MYO2 deletions were generated with the QuikChange® Site-Directed Mutagenesis Kit (Stratagene) using a pBluescript plasmid (NLC15) containing the 1.6-kb EcoRI fragment of pRS413-MYO2 (Catlett and Weisman, 1998). For pGBD-C1-myo2 11131518, the primers SDP 7F (5'-GGGTCACGAGCATAGCTGAAGCATATTTATCACTCC-3') and SDP 7R (5'-GGAGTGATAAATATGCTTCAGCTATGCTCGTGACCC-3') were used to generate a stop codon at amino acid 1518. For pGBD-C1-myo2 11131574 12971307, the primers SDP 9F (5'-GGTCACAGAACTAAAGGATATTTGGCTGAAGAAATTGCAG-3') and SDP 9R (5'-CTGCAATTTCTTCAGCCAAATATCCTTTAGTTCTGTGACC-3') were used. The
1.3-kb StuI and ClaI fragments from the above plasmids were each ligated into pGBD-MYO2 11131574 missing the corresponding region. For pGBD-myo2 11131568, the primers SDP 8F (5'-GACCTTGTTGCCCAATAAGTCGTTCAAGACGG-3') and SDP 8R (5'-CCGTCTTGAACGACTTATTGGGCAACAAGGTC-3') were used to generate a stop codon at amino acid 1568. The
1.5-kb EcoRI and ClaI fragment was subcloned into pGBD-C1-myo2 11131574 missing the corresponding region.
Random mutagenesis of VAC17
To isolate VAC17 suppressors of myo2-N1304S, PCR mutagenesis was performed using Taq DNA polymerase (Boehringer). Full-length VAC17 was PCR amplified from pRS415-VAC17 using the primer set TF1v (see above) and TF2v (5'-AAAACTGCAGAAGATGGCACCCGAGTCTAG-3'). LWY6631 (pGAL-PEP4 -URA) was cotransformed with the mutated VAC17 PCR products and a VAC17 plasmid cut with MscI and SphI to remove most of VAC17.
In vivo labeling of organelles
Yeast vacuoles were labeled with N-(3-triethylammoniumpropyl)-4(6(4(diethylamino)phenyl)hexatrienyl) pyridium dibromide (FM464; Molecular Probes, Inc.) as described previously (Catlett et al., 2000). Low and high copy VAC17 were expressed from the pRS416 and pRS426 plasmids, respectively (Tang et al., 2003). Nuclei were observed with 4' DAPI dihydrochloride hydrate (Sigma-Aldrich) as described previously (Sherman et al., 1986).
Immunofluorescence labeling and vacuole purification
Indirect immunofluorescence was performed essentially as described previously (Hill et al., 1996; Catlett et al., 2000). Goat anti-Myo2p tail antiserum (Catlett, 2000) was affinity purified as previously described (Reck-Peterson et al., 1999). Fixed cells were incubated with affinity-purified goat anti-Myo2p tail antibody (1:200), followed by Alexa 488-donkey antigoat IgG (1:200). Vacuole membranes were labeled with mouse antiyeast v-ATPase 60-kD subunit (1:200), followed by Rhodamine red X-donkey antimouse IgG (1:200). Secondary antibodies and antiyeast v-ATPase were purchased from Molecular Probes, Inc.
Vacuoles were isolated on a Ficoll flotation gradient as described previously (Catlett and Weisman, 1998), except 40 µM chymostatin, 10 mM DTT, 1x complete, EDTA-free protease inhibitor cocktail (Roche), and 1x protease inhibitor cocktail (Sigma-Aldrich), were added to the cell suspension and Ficoll gradient layers. After removal from the gradient, vacuoles were washed once in the gradient buffer and collected by centrifugation at 13,000 g for 10 min.
Fluorescence microscopy
Cells were viewed with a microscope (Axioscope 2, Carl Zeiss MicroImaging, Inc.) equipped for epifluorescence, and images were captured using an RT Spot camera (Diagnostic Instruments, Inc.) controlled by MetaMorph® Imaging Series 4.5 software (Universal Imaging Corporation).
Confocal images were obtained with a laser scanning confocal microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.). For each field, a z-series of 0.3-µm slices was scanned and projected to generate a single image. The data was exported as 8-bit TIFF files and processed using Adobe Photoshop®.
Western blot analysis
SDS-PAGE and Western blot analysis were performed after standard procedures. Primary and secondary antibodies were used at the following concentrations: affinity-purified goat anti-Myo2p tail (1:2,000), HRP-donkey antigoat IgG (1:5,000; Jackson ImmunoResearch Laboratories), mouse antiyeast v-H+-ATPase 100-kD subunit (1:5,000; Molecular Probes, Inc.), and HRP-goat antimouse IgG (1:5,000; Molecular Probes, Inc.). For Fig. 3 E, the concentrations of primary and secondary antibodies were goat anti-Myo2p tail (1:10,000) and HRP-donkey antigoat IgG (1:10,000). HRP activity was detected using ECL (Amersham Biosciences).
![]() |
Acknowledgments |
---|
This work was supported by the National Institutes of Health (grant RO1 GM62261), the National Science Foundation (MCB 96-00867), and an Established Investigator Award from the American Heart Association (0140233N; to L.S. Weisman). N.L. Catlett was supported in part by a National Institute on Aging Grant (T32 AG 00214) awarded to The Interdisciplinary Research Training Program on Aging, University of Iowa.
Submitted: 24 October 2002
Revised: 17 January 2003
Accepted: 23 January 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beach, D.L., J. Thibodeaux, P. Maddox, E. Yeh, and K. Bloom. 2000. The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast. Curr. Biol. 10:14971506.[CrossRef][Medline]
Bonangelino, C.J., N.L. Catlett, and L.S. Weisman. 1997. Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology. Mol. Cell Biol. 17:68476858.[Abstract]
Catlett, N.L. 2000. Role of a yeast myosin-V in movement of the vacuole and other cargoes. In Department of Biochemistry. University of Iowa, Iowa City. 135.
Catlett, N.L., and L.S. Weisman. 1998. The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth. Proc. Natl. Acad. Sci. USA. 95:1479914804.
Catlett, N.L., J.E. Duex, F. Tang, and L.S. Weisman. 2000. Two distinct regions in a yeast myosin-V tail domain are required for the movement of different cargoes. J. Cell Biol. 150:513526.
Fukuda, M., and T.S. Kuroda. 2002. Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa, and actin. J. Biol. Chem. 277:4309643103.
Gomes de Mesquita, D.S., H.B. van den Hazel, J. Bouwman, and C.L. Woldringh. 1996. Characterization of new vacuolar segregation mutants, isolated by screening for loss of proteinase B self-activation. Eur. J. Cell Biol. 71:237247.[Medline]
Govindan, B., R. Bowser, and P. Novick. 1995. The role of Myo2, a yeast class V myosin, in vesicular transport. J. Cell Biol. 128:10551068.[Abstract]
Hill, K.L., N.L. Catlett, and L.S. Weisman. 1996. Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae. J. Cell Biol. 135:15351549.[Abstract]
Hoepfner, D., M. van den Berg, P. Philippsen, H.F. Tabak, and E.H. Hettema. 2001. A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae. J. Cell Biol. 155:979990.
Hume, A.N., L.M. Collinson, C.R. Hopkins, M. Strom, D.C. Barral, G. Bossi, G.M. Griffiths, and M.C. Seabra. 2002. The leaden gene product is required with Rab27a to recruit myosin Va to melanosomes in melanocytes. Traffic. 3:193202.[CrossRef][Medline]
James, P., J. Halladay, and E.A. Craig. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 144:14251436.
Johnston, G.C., J.A. Prendergast, and R.A. Singer. 1991. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J. Cell Biol. 113:539551.[Abstract]
Karcher, R.L., J.T. Roland, F. Zappacosta, M.J. Huddleston, R.S. Annan, S.A. Carr, and V.I. Gelfand. 2001. Cell cycle regulation of myosin-V by calcium/calmodulin-dependent protein kinase II. Science. 293:13171320.
Lapierre, L.A., R. Kumar, C.M. Hales, J. Navarre, S.G. Bhartur, J.O. Burnette, D.W. Provance, Jr., J.A. Mercer, M. Bahler, and J.R. Goldenring. 2001. Myosin vb is associated with plasma membrane recycling systems. Mol. Biol. Cell. 12:18431857.
Melkonian, K.A., A.G. Ostermeyer, J.Z. Chen, M.G. Roth, and D.A. Brown. 1999. Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J. Biol. Chem. 274:39103917.
Nagashima, K., S. Torii, Z. Yi, M. Igarashi, K. Okamoto, T. Takeuchi, and T. Izumi. 2002. Melanophilin directly links Rab27a and myosin Va through its distinct coiled-coil regions. FEBS Lett. 517:233238.[CrossRef][Medline]
Nau, J.J., K.R. Summers, A.M. Galbraith, S.A. Bullard, and R.E. Malone. 1997. Isolation of early meiotic recombination genes analogous to S. cerevisiae REC104 from the yeasts S. paradoxus and S. pastorianus. Curr. Genet. 31:714.[CrossRef][Medline]
Ortiz, D., M. Medkova, C. Walch-Solimena, and P. Novick. 2002. Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast. J. Cell Biol. 157:10051015.
Provance, D.W., T.L. James, and J.A. Mercer. 2002. Melanophilin, the product of the leaden locus, is required for targeting of myosin-Va to melanosomes. Traffic. 3:124132.[CrossRef][Medline]
Reck-Peterson, S.L., P.J. Novick, and M.S. Mooseker. 1999. The tail of a yeast class V myosin, Myo2p, functions as a localization domain. Mol. Biol. Cell. 10:10011017.
Rossanese, O.W., C.A. Reinke, B.J. Bevis, A.T. Hammond, I.B. Sears, J. O'Connor, and B.S. Glick. 2001. A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae. J. Cell Biol. 153:4762.
Schott, D., J. Ho, D. Pruyne, and A. Bretscher. 1999. The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting. J. Cell Biol. 147:791808.
Sherman, F., G.R. Fink, and J.B. Hicks. 1986. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 153 pp.
Singer, J.M., G.J. Hermann, and J.M. Shaw. 2000. Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions. Genetics. 156:523534.
Tang, F., E.J. Kauffman, J.L. Novak, J.J. Nau, N.L. Catlett, and L.S. Weisman. 2003. Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole. Nature. 10.1038/nature01453.
Thomas, J.H., and D. Botstein. 1986. A gene required for the separation of chromosomes on the spindle apparatus in yeast. Cell. 44:6576.[Medline]
Vida, T.A., T.R. Graham, and S.D. Emr. 1990. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J. Cell Biol. 111:28712884.[Abstract]
Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. Fusion of docked membranes requires the armadillo repeat protein Vac8p. J. Biol. Chem. 276:3513335140.
Winston, F. 1990. Mutagenesis of yeast cell. Current Protocols in Molecular Biology. F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman, and K. Struhl, editors. Greene Publishing Associates and Wiley-Interscience, New York. 13.3.113.3.4.
Wu, X., F. Wang, K. Rao, J.R. Sellers, and J.A. Hammer III. 2002a. Rab27a is an essential component of melanosome receptor for Myosin va. Mol. Biol. Cell. 13:17351749.
Wu, X.S., K. Rao, H. Zhang, F. Wang, J.R. Sellers, L.E. Matesic, N.G. Copeland, N.A. Jenkins, and J.A. Hammer III. 2002b. Identification of an organelle receptor for myosin-Va. Nat. Cell Biol. 4:271278.[CrossRef][Medline]
Yin, H., D. Pruyne, T.C. Huffaker, and A. Bretscher. 2000. Myosin V orientates the mitotic spindle in yeast. Nature. 406:10131015.[CrossRef][Medline]
Zacharias, D.A., J.D. Violin, A.C. Newton, and R.Y. Tsien. 2002. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 296:913916.