Figure 3.

Gga2p is required for cell-free TGN-to-PVC transport of Kex2p and K-V. (A) Schematic depiction of MAY17 construction (see Materials and Methods) and expression of Gga2p-myc. Whole-cell lysates were prepared from JBY209 (GGA1 GGA2) and MAY17 (gga1 GGA2::13xmyc). Proteins were resolved by SDS-PAGE (10% acrylamide body gel) and analyzed by immunoblotting using anti-c-myc. The position of Gga2p-myc and molecular mass size markers are indicated. (B) GGA2::13myc expresses Gga2-13myc and functionally replaces Gga2p. JBY209 (GGA1 GGA2), MAY14 (gga1 gga2), and MAY17 (gga1 GGA2::13myc) were analyzed for the Vps phenotype (CPY secretion; see Materials and Methods). (C) MSS was prepared from strains JBY209 (GGA1 GGA2) and MAY17 (gga1 GGA2::13myc) expressing Kex2p, K-V or PSHA. Donor and acceptor MSS samples were combined and preincubated with 12 μg of anti-c-myc for 1 h on ice. Cell-free transport reactions were then carried out (20 min at 30°C). Extents of transport observed for controls (no anti-c-myc) were 11.3%, JBY209 (Kex2p); 13.8%, MAY17 (Kex2p); 10.4%, JBY209 (K-V); and 11.9%, MAY17 (K-V), adjusted to 100% in the figure. Experimental values (+ anti-c-myc) are shown as percentage of the control values. (D) Anti-c-myc recognizes Gga2p-myc during the course of the reaction. An aliquot of each reaction shown in C was incubated with protein A-Sepharose for 1 h at 4°C. Protein A-Sepharose pellets were subsequently washed and solubilized in SDS-PAGE sample buffer. Eluted samples were resolved by SDS-PAGE and analyzed by immunoblotting using anti-c-myc. (E) Titration of anti-c-myc in transport reactions. Donor and acceptor MSS samples were combined and preincubated with the indicated amounts of anti-c-myc for 1 h on ice. Transport reactions were then carried out (20 min at 30°C). Data are expressed as percentage of control reactions as described in (C). Extents of transport in control (no anti-c-myc) reactions were 11.2% MAY17 (Kex2p) and 11.9% MAY17 (K-V).