QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 20 July 2004
doi: 10.1242/jcs.01239

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yu, X. Articles by Cai, M. Search for Related Content PubMed PubMed Citation Articles by Yu, X. Articles by Cai, M. Social Bookmarking                         What's this?

The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p

Institute of Molecular and Cell Biology, National University of Singapore, 61 Biopolis Drive, Proteos, Singapore 138673, Rep. of Singapore

View larger version (69K): [in a new window]   Fig. 1. Vps1p is required for normal actin cytoskeleton organization. (A) Actin cytoskeleton in several yeast mutants at different temperatures. Strains (YMC451) vps1, (YMC452) dnm1, (YMC453) mgm1, (YMC454) vps26 and wild-type W303-1-B (W303) cultured to mid-log phase in YEPD at 24°C were shifted to 30°C or 37°C for 3 hours before being subjected to actin staining (bar, 5 µm). (B) Quantitative illustration of the populations of vps1 and W303 with actin abnormalities. Actin depolarization shown in the left panel was calculated only in budded cells with small- and medium-sized buds, and the actin aggregation shown in the right panel was based on the total cell population. (C) The abnormal budding pattern and chitin deposition in vps1 cells. Strains YMC456 (vps1, diploid) and the wild type YMC455 (W303, diploid) were grown in YEPD to log phase at 24°C. Aliquots of the cultures were then shifted to either 30°C or 37°C for 3 hours before being subjected to Calcofluor staining. The budding patterns and chitin distributions of cells incubated at different temperatures were scored and shown in Table 4 (bar, 5 µm). (D) Prolonged half-life of Ste3p in vps1 cells. YMC451 (vps1) () and the wild-type W303-1-B (W303) () cells that contain GAL1-STE3-EGFP were induced to express Ste3p-GFP for 90 minutes in 2% of galactose at 25°C, followed by addition of 3% glucose to repress the expression. The cultures were immediately shifted to 30°C or 37°C. The amount of Ste3p-EGFP at indicated times after glucose addition in W303 and vps1 cells were detected by western blotting (lower panels). The levels of Ste3-EGFP were quantified using a densitometer and plotted in upper panels.

View larger version (62K): [in a new window]   Fig. 2. The putative GTPase mutants of vps1 affect actin cytoskeleton. (A) Alignment of the GTPase domain and the GED domain from mammalian dynamin-1 with three yeast dynamin-related proteins. Numbers on the top indicate the amino acid positions of Vps1p. Underlined in the GTPase domain are the residues that were mutated. (B) Temperature sensitivities of the vps1 cells containing various GTPase mutants of vps1. Strains YMC457 (vector/vps1), YMC458 (Vps1WT/vps1), YMC459 (vps1K42E/vps1), YMC460 (vps1S43N/vps1) and YMC461 (vps1G315D/vps1) cultured at 24°C were first diluted to similar density from which further serial dilutions were made. We spotted 5 µl of each dilution onto the YEPD plates and incubated at indicated temperature for one day. (C) LAT-A sensitivity of the vps1 and GTPase mutants. Cells shown in (B) were cultured in YEPD at 30°C and subjected to LAT-A halo assay as described in Materials and Methods. The bar chart in the right shows the quantified result. (D) The uracil-uptake assays in the YMC458 (VPS1) (), YMC457 (vps1) () and YMC459-461 (vps1K42E, vps1S43N and vps1G315D) (,,) at both 30°C (left) and 37°C (right).

View larger version (66K): [in a new window]   Fig. 3. The actin defects caused by over-expression of the GTPase domain mutants of vps1. (A) Over-expression of the GTPase domain mutants caused abnormal actin structures. Strains integrated with the following GAL1-driven vps1 constructs, YMC463 (Gal-Vps1WT), YMC464 (Gal-vps1K42E), YMC465 (Gal-vps1S43N) and YMC466 (Gal-vps1G315D), as well as YMC462 (vector), were cultured to mid-log phase in the presence of 2% raffinose (strain name in brackets, see Table 1). Galactose was added to 2% to induce the expression of the vps1 mutants for 4 hours at 30°C. Cells were then collected for actin staining (Bar, 5 µm). (B) Over-expression of the GTPase mutants caused lethality in wild-type cells. The strains as described in (A) were grown on glucose-containing plates and replica-plated onto galactose plates and incubated at 37°C for 1 day before being photographed. (C) Cells over-expressing the vps1 GTPase mutants are hypersensitive to LAT-A. The strains as described in (A) were plated on the galactose-containing medium at 30°C and the LAT-A with the indicated concentration was applied (left panel). A summary of the halo assay is shown in the graph at the right.

View larger version (67K): [in a new window]   Fig. 4. Synthetic lethality between vps1 and alleles of sla1 with actin defects. (A) The viabilities of various mutants after their SLA1-containing plasmid pTHY918 was lost on a 5-FOA plate: strainsYMC467 (sla1), YMC468 (sla1 mgm1), YMC469 (sla1 dnm1) and YMC470 (sla1 vps1) grown on SC-Ura plate (left) at 24°C were replica-plated onto a 5-FOA plate (right) and incubated at 24°C for 2 days. (B) The schematic structures of Sla1p and its deletion constructs to be used in the following experiments. (C) The test of the strain YMC470 (sla1 vps1) to lose the SLA1-containing plasmid pTHY918 in the presence of various sla1 deletion constructs: pRS314 (vector, lane 1), pYGS203 (sla13rdSH3, lane 2), pTHY1114 (sla1all SH3, lane 3), pTHY1115 (sla1SR, lane 4) and pTHY1089 (Sla1FL, lane 5).

View larger version (31K): [in a new window]   Fig. 5. Physical interaction between Vps1p and Sla1p. (A) Co-immunoprecipitation of Vps1p and Sla1p. Equal amounts of protein extracts prepared from W303 containing GAL1-myc-VPS1 (lanes 3, 6 and 7), HA-SLA1 (pTHY1108, lanes 1, 4 and 9), or both (lanes 2 and 8), were subjected to anti-HA or anti-Myc immunoprecipitation (IP) and followed by SDS-PAGE and immunoblotting (IB). Vps1-Myc was present in the immunoprecipitates of HA-Sla1p from the co-transformants (lane 5). (B) The physical association between the mutated Vps1p and Sla1p. Strains expressing Vps1p or vps1 GTPase mutants (YMC 458-461) were transformed with HA-SLA1 (pTHY1108) and the vector (pRS314), respectively. Equal amounts of protein extracts prepared from the above strains were subjected to anti-HA or anti-Myc immunoprecipitation (IP) and followed by SDS-PAGE and immunoblotting using anti-Myc antibody.

View larger version (87K): [in a new window]   Fig. 6. Vps1p is required for the cell cortex and polarized localization of Sla1p. The subcellular localization of Sla1p and actin morphology were altered in vps1 and the vps1 GTPase mutants. The genomic copy of SLA1 in strains YMC457 (vps1), YMC458 (vps1 with wild-type VPS1, Vps1WT) and YMC459-461 (vps1 GTPase mutants, vps1K42E, vps1S43N and vps1G315D, respectively), was tagged with GFP at its C terminus and the resulting strains were incubated at 30°C to the exponential phase followed by actin staining. The actin organization and the subcellular localization of Sla1p from vps1 and the vps1 GTPase mutants were visualized under Leica microscopy (bar, 5 µm).

View larger version (92K): [in a new window]   Fig. 7. The effects of over-expression of the vps1 C-terminal region. (A) The structures of Vps1p and its deletion constructs used in the experiments below. Numbers on the top indicate the amino acid positions. (B) Over-expressions of the C-terminal region of Vps1p caused cell death at 37°C. Wild-type cells containing various deletion constructs of VPS1 (pGAL1-VPS1C1C4, pGAL1-VPS1-N1), as well as the full length VPS1 (pGAL1-VPS1) were grown on glucose containing medium and replicated to galactose plates and incubated at 37°C for one day. (C) The localization of Sla1p was affected by the over-expression of the C-terminal regions of Vps1p. The endogenous copy of SLA1 in wild-type cells was tagged with YFP tag at its C-terminus and the resulting integrant was transformed with various deletion constructs of VPS1 indicated in A. The resulting transformants were cultured in raffinose-containing media to the mid-log phase before galactose was added and incubated for another 3 hours at 30°C. The localization of the various domains of Vps1p along with that of Sla1p was examined under Leica microscopy. Note the partial co-localization of Sla1p with Vps1p and its domains in 1, 4 and 6 (bar, 5 µm). (D) The strains as in (B) were analyzed for their sensitivities to LAT-A. The cells were plated on the galactose-containing medium at 30°C and the LAT-A with the indicated concentrations was applied (left panel). The results were summarized graphically in the right panel.

View larger version (52K): [in a new window]   Fig. 8. The defects of different vps1 mutants in vacuolar protein sorting. (A) Abnormal secretion of CPY in the vps1 and the GTPase mutants. Strains as indicated (YMC457-461) were cultured in YEPD media to saturation at 24°C. The cell densities from these cultures were normalized and 5 µl of them was spotted onto YEPD plates. After 2 days at 24°C, cells were replica-plated onto YEPD plates, overlaid with a nitrocellulose membrane, and incubated overnight at 30°C. The membranes were then probed with a monoclonal antibody against CPY. (B) Over-expression of the vps1 GTPase mutants caused the sorting defects of CPY in wild-type cells. Strains of YMC462-466 were first cultured in raffinose media at 24°C for 2 days. Cell numbers were normalized and 5 µl of them was spotted onto galactose-containing plates. After an overnight incubation at 24°C, cells were replica-plated onto refresh galactose plates, overlaid with a nitrocellulose membrane and incubated overnight at 30°C. The membranes were probed with a monoclonal antibody against CPY. (C) Over-expression of the C-terminal region of Vps1p resulted in the secretion of CPY in wild-type cells. Strains as indicated were assayed for CPY sorting as described in (B).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter