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. 2001 Dec;21(23):7981–7994. doi: 10.1128/MCB.21.23.7981-7994.2001

FIG. 2.

FIG. 2

The gga1Δ gga2Δ mutant is defective for α-factor maturation and shows a decrease in Kex2p levels. (A) Secretion of mature α-factor in the gga1Δ gga2Δ mutant. Wild-type (MATα), gga1Δ gga2Δ (MATα) mutant, and wild-type (MATa) (negative control) cells were serially diluted to concentrations indicated, and equal amounts were spotted on rich medium (YEPD) containing a freshly spread lawn of α-factor-supersensitive mutant strain RC634 (MATa sst1-3). The agar plate was then incubated at 30°C to assess α-factor secretion of test strains as indicated by relative growth inhibition of sst1-3 mutant cells (halo). (B) Processing of α-factor in the gga1Δ gga2Δ mutant. Strains described for panel A were pulse-labeled at 25°C for 7.5 min as described in Materials and Methods. Various α-factor Golgi-processing intermediates and mature α-factor were immunoprecipitated with anti-α-factor antibodies, resolved by SDS-PAGE (4 to 20% acrylamide gels), and analyzed by fluorography. pαf (pro-α-factor) denotes a range of high-molecular-mass α-factor intermediates (∼90 to 150 kDa) containing the pro-region and Golgi complex-derived, complex glycosylation. unglyc and core refer to unglycosylated (∼20-kDa) and ER-localized, core-glycosylated (∼26-kDa) forms of pro-α-factor, respectively. inter-αf and mαf represent a range of low-molecular-mass α-factor intermediates (probably the α-factor tetrapeptide in various states of processing but with the prosequence removed) (∼6 to 8 kDa) and the 13-aa terminally processed (mature-secreted) form (∼2 kDa), respectively. Relative positions of molecular mass markers are indicated on the right. (C) Steady-state levels of Kex2p in the gga1Δ gga2Δ mutant. Whole-cell lysates were prepared from wild-type, gga1Δ gga2Δ, and vps1 cells expressing epitope-tagged Kex2p. Proteins were resolved by SDS-PAGE (8% acrylamide gels) and analyzed by immunoblotting using anti-HA and anti-Kar2p (to assess relative protein loading) antibodies as described in Materials and Methods. Positions of Kex2p and Kar2p and molecular mass size markers are indicated.