Figure 3.
Cbk1 is required for secretion of invertase, Bgl2 endo-β-1,3-glucanase, and other proteins. (A) Invertase secretion is diminished in conditional cbk1-8 cells. The ratio of secreted invertase/total invertase is plotted for wild-type cells (BY4741), cbk1-8 cells (FLY2661), and the conditional exocyst mutant exo84-121 (GY2479). See Materials and Methods for details. (B) The cell wall protein Bgl2 accumulates inside cbk1-8 and exo84-121 cells at 37°C. Top, immunoblot of Bgl2 in the internal fraction (cytoplasmic) of wild type, cbk1-8, and exo84-121 cells shifted to restrictive temperature. Middle, immunoblot of Bgl2 from the external (solubilized cell wall) fraction of cells. Bottom, immunoblot of the internal fractions was stripped and reprobed with anti-ADH (Abcam) as a protein loading. bgl2Δ cells (FLY2574) were used as a negative control. (C) Autoradiogram of media secretion assays for wild-type cells (BY4741), cbk1-8 cells (FLY2704), cog1Δ cells (FLY2764), ace2Δ cells (FLY1632), ssd1Δ cells (FLY2184), and cbk1Δ ssd1Δ cells (FLY2293). Proteins secreted into the media of [35S]methionine-labeled cells were TCA precipitated, separated on protein gels, and processed for autoradiography. The brackets denote several proteins that seem to be hypoglycosylated in cbk1-8 cells. Hsp150 is noted and the arrowhead below Hsp150 points to a protein whose expression and/or secretion is dependent on Cbk1 and Ace2. The two arrows on the right point to a protein that seems to split into two bands in cbk1Δ ssd1Δ double mutant cells.