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First published online 14 February 2006
doi: 10.1242/jcs.02849

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The polarity-establishment component Bem1p interacts with the exocyst complex through the Sec15p subunit

¹ Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA
² Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

View larger version (27K): [in a new window]   Fig. 1. The bem1-3 growth defect is suppressed by both SEC15 and SEC4 multi-copy plasmids. S. cerevisiae bem1-3 cells (NY1444) carrying 2 µ SEC15, 2 µ SEC4 or 2 µ BEM1 plasmids (pNB192, pNB142 and pNB1227, respectively) were spotted onto YPD plates along with bem1-3 cells carrying no plasmid. The plates were incubated at 25°C, 30°C, 34°C and 37°C for 3 days and scanned.

View larger version (38K): [in a new window]   Fig. 2. The C-terminus of Sec15p physically interacts with Bem1p, whereas the N-terminus of Sec15p is required for the interaction with Sec10p. Cell growth, yeast lysate preparations, GST pull-down and western blotting procedures were as described in the Materials and Methods. (A) Schematic diagram of Sec15p constructs employed in this study. (B) Sec15-Bem1 complexes. Glutathione-Sepharose pull-down products from lysates of cells expressing GST-fusion products of Sec15 (Sec15 GST; NY2559) and Sec15-1 (sec15-1 GST; NY2560), and GST (NY2561), were analyzed by standard SDS-PAGE and western blotting with the indicated antibodies. (C) Glutathione-Sepharose pull-down products from lysates of cells expressing GST-fusion products of the Sec15p constructs described in A were analyzed by standard SDS-PAGE and western blotting with the indicated antibodies. The purified Sec15-GST constructs were resolved on an SDS-PAGE gel and the gel was analyzed by western blotting using anti-GST antibodies. Lysates were loaded to indicate that there were similar amounts of Sec10p present in all lysates although only the full-length Sec15p containing the extreme N-terminal region can pull-down Sec10p. (D) Diagram of the protein-protein interactions of Sec15.

View larger version (49K): [in a new window]   Fig. 3. Bem1p can interact with the exocyst complex through Sec15p. (A) Purified recombinant GST-Bem1p (GST-Bem1) and GST were resolved by SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. Proteins of the predicted molecular masses were observed. (B) Exocyst complex purified from yeast strain NY2521 was resolved by SDS-PAGE and the gel was silver stained to visualize the subunits. (C) GST-Bem1p (GST-Bem1) or GST immobilized on glutathione-Sepharose beads was incubated with the purified exocyst complex as described in the Materials and Methods. Lanes 1 and 2 represent 2.5% and 5%, respectively, of the exocyst complex used in the binding reactions. The beads were washed four times with the binding buffer and the bound proteins were eluted with SDS-PAGE sample buffer. The eluates were then resolved by SDS-PAGE, and western blot analysis was performed using anti-HA (to detect Sec5-3xHA), anti-Sec15 and Sec10 antibodies.

View larger version (32K): [in a new window]   Fig. 4. The C-terminus of Sec15p directly interacts with Bem1p, and the N-terminus of Bem1p is necessary and sufficient for Sec15p interaction. (A) Schematic diagram of recombinant proteins used in this study. Several proteins that have been reported to interact with specific domains of Bem1p are indicated. SH3, Src-homology 3; PX, Phox homology domain; PB1, Phox and Bem1 domain; 6xHis, 6-histidine tag. (B) The Sec15p C-terminal region can directly interact with Bem1p. The C-terminus of Sec15 (residues 557-910) was fused to GST (GST-Sec15557-910), purified and immobilized on the resin. Bem1p was fused to a 6xHis tag and purified from baterial lysates. These proteins were used in an in vitro binding assay as described in the Materials and Methods. The panel on the left shows purified recombinant GST-Sec15557-910 and GST constructs resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Proteins of appropriate molecular masses were observed. The C-terminal fragment of Sec15 immobilized to glutathione-Sepharose beads was incubated with bacterially purified Bem1p. The beads were then washed and the bound proteins were resolved by SDS-PAGE and detected by western blot analysis using anti-Bem1p serum. (C) The interaction with Sec15p requires the N-terminal region of Bem1p. The panel on the left shows N-terminally truncated alleles of Bem1p expressed in bacteria. For binding studies, crude bacterial lysates containing these truncated constructs were incubated with GST-Sec15557-910 immobilized on glutathione beads. The beads were washed and the bound products were resolved by SDS-PAGE and detected by western blot analysis using anti-His monoclonal antibody. (D) The N-terminal 138 residues are necessary and sufficient for mediating the Sec15p interaction. The crude bacterial lysates containing the minimal 138 amino acids at the N-terminus of Bem1 were incubated with GST-Sec15557-910 on glutathione beads and the binding assay was performed in similar fashion to panel C.

View larger version (49K): [in a new window]   Fig. 5. A fraction of Sec15p localizes in an actin-independent fashion. Yeast cells were grown in SC-Ura overnight to early log phase. For GFP visualization, cells were harvested, fixed in methanol, permeabilized in acetone, rehydrated in PBS, and imaged in both fluorescence and differential interference contrast (DIC) modes. Actin polarization was monitored by staining cells with FITC-conjugated phalloidin. (A) An overnight culture of act1-3 cells carrying Sec15-GFP as the sole copy of Sec15p (Sec15GFP; NY2556) was shifted to 37°C for 90 minutes (or incubated at 20°C) prior to methanol fixation. The localization was examined by direct fluorescence microscopy and the proportion of cells containing a polarized GFP signal was scored (right). At least 200 cells were scored in each strain. (B) Wild-type yeast cells expressing GFP-tagged Sec15 (Sec15GFP; NY2557) were grown to early log phase and treated either with DMSO alone or with 200 µM latrunculin A (LatA) in DMSO. Following treatment for 15 minutes, cells were rapidly fixed by methanol/acetone and prepared for microscopy. The localization was examined by direct fluorescence microscopy and the proportion of cells containing a polarized GFP signal was scored (right). At least 200 cells were scored in each strain. (C) The localization of Sec15-GFP (Sec15GFP) in wild-type cells (NY2557) exiting G0 at 25°C in the absence or presence of latrunculin A was examined at time points of 1, 2 and 3.5 hours. (D) The localization of Sec8-GFP (Sec8GFP; NY2558) in wild-type cells exiting G0 was examined and the cells were observed after 3.5 hours of incubation in the absence or presence of latrunculin A (Lat-A). (E) Quantification of the GFP fluorescence to assess the percentage of cells incubated in the absence or presence of Lat-A that exhibited polarized Sec15-GFP (Sec15GFP) or Sec8-GFP (Sec8GFP) proteins. At least 200 cells were scored in each strain.

View larger version (42K): [in a new window]   Fig. 6. bem1SH3-1 mutant cells have a mild polarity defect and the polarized localization of Sec15-GFP is disrupted in bem1SH3-1 mutant cells. (A) The percentage of yeast mother cells with oval-shaped wild-type versus round morphology was determined as described in the Materials and Methods for wild-type (Wt) and bem1SH3-1 mutant (bem1-SH3-1) cells. For each dataset, the yeast strains were grown in parallel and DIC images were taken to measure ratios (n) of length to width. The average and s.d. were calculated from two independent datasets, and at least 200 cells were measured per strain and dataset. (B) bem1SH3-1 mutant (bem1-SH3-1; NY2568) and wild-type (NY2557) strains containing Sec15-GFP were grown overnight at 25°C to early log phase and treated either with DMSO alone or with 200 µM latrunculin A (Lat-A). After 15 minutes of treatment, cells were rapidly fixed by methanol/acetone and mounted for microscopy. The localization was examined by direct fluorescence microscopy. Examples of cells with a small bud are marked with large arrows and those with a large bud are marked with smaller arrows. Unbudded cells are marked with arrowheads. (C) Wild-type (Wt) and bem1SH3-1 (SH3-1) cells were categorized based upon their pattern of Sec15-GFP polarized localization – the presumptive bud, bud tip or the bud neck. The graph depicts the percentage of the population within each category. At least 200-300 cells were scored in each strain. (D) The localization of Sec15-GFP in synchronized wild-type and bem1SH3-1 (bem1-SH3-1) cells released from G0 at 25°C in the absence or presence of Lat-A was examined at time points of 0 and 2 hours. (E) Quantification of GFP fluorescence, used to assess the percentage of cells that exhibited polarized Sec15-GFP in the presence or absence of Lat-A treatment. At least 200 cells were scored in each strain.

View larger version (14K): [in a new window]   Fig. 7. Sec15-GFP recovers from photobleaching more slowly in the presence of latrunculin A. (A) Strain NY2445 containing Sec15-GFP (Sec15GFP) expressed from a fusion construct integrated as the sole copy of SEC15 at the SEC15 locus was grown overnight and used in photobleaching experiments as described in the Materials and Methods. Two representative recovery curves generated from photobleaching Sec15-GFP in the presence of DMSO alone or DMSO + 200 µM latrunculin A (Lat A) are shown. (B) In order to determine how many modes of action were causing Sec15-GFP recovery after photobleaching, the natural log of each point from the example recovery curve from part A was plotted against time. The straight line through the data indicates that only one mode of recovery was detectable for each. (C) The half-time of recovery for specimens incubated with Lat A was compared with the half-time of recovery for untreated specimens and found to be approximately four times longer; 90±16 seconds (n=13) versus 22±5 seconds (n=15).

View larger version (44K): [in a new window]   Fig. 8. Sec15-1–GFP mislocalizes as does Sec8-GFP in a sec15-1 background. (A) GFP-tagged sec15-1 (sec15-1 GFP; NY2565) or SEC15 (Sec15 GFP; NY2557) cells were shifted to restrictive temperature (37°C) or grown at permissive temperature (25°C) for 1.5 hours and processed for GFP fluorescence detection. Cells were visualized by direct fluorescence microscopy. (B) The proportion of cells containing a polarized GFP signal from the experiment shown in panel A was scored. At least 200 cells were scored in each strain. (C) Cells expressing GFP-tagged Sec8 (Sec8GFP), either in a wild-type (NY2558) or sec15-1 (sec15-1; NY2566) background were shifted to restrictive (37°C) or grown at permissive (25°C) temperature for 1.5 hours and processed for the direct GFP fluorescence detection. The localization was visualized by direct fluorescence microscopy. (D) The proportion of cells containing a polarized GFP signal from the experiment shown in panel C was scored. At least 200 cells were scored in each strain.