Figure 1.
EM immunogold localization of Cdc42p in sec6-4ts cells.
EM immunogold localization of Cdc42p (A) or Rho1p (D) in log-phase sec6-4 cells shifted to 37°C for 60 min. B and E) Distribution of gold particles among various membrane compartments within buds of the same preparations shown in (A) and (D) when probed for Cdc42p (B; n = 37 cells) or Rho1p (E; n = 33 cells). PM, plasma membrane; V, 80–100 nm diameter vesicles; ER, endoplasmic reticulum; ER-PM, endoplasmic reticulum and/or plasma membrane; ER-V, endoplasmic reticulum and/or vesicles; V-PM, vesicles and/or plasma membrane; UN (unassigned), membrane compartment of ambiguous identity. C and F) Average number of gold particles within the bud and mother of the same cell, scored on the same section, standardized to an area of 1 um2. Error bars indicate SEM. Cells and sections scored in (C and F) are the same as those scored in (B and E), respectively.
Figure 2.
EM immunogold localization of Cdc42p in wild-type cells.
A) EM immunogold localization of Cdc42p in log-phase wild-type cells (DDY904) grown at 25°C. Panels in top row show the buds of the corresponding cells in the bottom row. Arrows indicate gold labeling of secretory vesicles. B) Distribution of gold particles among various membrane compartments within buds of the same preparations shown in (A; n = 36 cells). Membrane compartment categories are the same as described for Figure 1B.
Figure 3.
Co-purification of Cdc42p with low density, Sec4p-marked vesicles.
A) Immunoblots of 100,000×g pellets of fractions collected from an 18–34% Nycodenz-sorbitol buoyant density gradient loaded with extracts prepared from equivalent OD600 units of sec6-4ts cells grown at 25°C or 37°C for 90 min. Each lane was loaded equivalent fraction volumes and, for the same probe, incubated with antibody of equivalent titer for the same amount of time. All blots are from the same fractionation, with the Sec4p and Bgl2p panels reprinted with permission from Alfaro et al. [37] © 2011 Traffic. All blots at an indicated temperature were prepared from the same fractions from the same gradient. B) Graph showing invertase activity across “37°C” buoyant density gradient shown in (A) and sucrose density across an unloaded gradient processed in parallel. Fraction densities were calculated based on a standard curve of Nycodenz-sorbitol concentration versus refractive index. C) Thin section electron micrographs showing vesicle association with a M500 Dynabead coated with mouse monoclonal anti-Sec4p antibody (left), but not with the same type of bead coated with mouse IgG (right), after incubation with a pooled, Sec4p-enriched, buoyant-density gradient fraction from sec6-4ts cells grown at 37°C for 90 min (comparable to fractions 11–14 in (A)). D) Immunoblots of a pooled, Sec4p-enriched, low-density gradient fraction (S), after incubation with Protein G-Dynabeads (P) coated with a monoclonal antibody against Sec4p or mouse IgG, and probed for plasma membrane marker Pma1p or post-Golgi secretory vesicle marker Sec4p. E) Immunoblots of pooled, Sec4p-enriched, low-density gradient fraction (S), after incubation with Dynabeads coated with a monoclonal antibody against Sec4p or Pma1p (P), probed for Cdc42p or Sec4p. In (D) and (E), the stoichiometric ratio of pellet to supernatant is 20∶1.