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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Biochem J. 2011 Jun 1;436(2):291–303. doi: 10.1042/BJ20101865

Figure 2. Depletion of WT Sss1p and expression of mutant tail-anchor proteins result in translocation defects and altered ER morphology.

Figure 2

(A) Depletion of WT Sss1p and accumulation of pre-Kar2p (pKar2p) and prepro-alpha-factor (pPαFactor) was determined by immunoblot analysis of whole cell lysates from an equal number of cell equivalents of yeast strain FKY173 grown in minimal media (minus adenine and histidine) supplemented with 0.2% amino acids and 2% glucose at 30°C. Yeast cells were grown for 10 hours and samples taken every two hours. Immunoblots were probed with antibodies to Kar2p, Sec61p, Porin, prepro-alpha-factor, and Sss1p and the relevant bands are identified to the right of the panels. (B) Expression levels of Sss1p and accumulation of pre-Kar2p and prepro-alpha-factor in yeast strain FKY173 transformed with an empty plasmid (vector) or with plasmids expressing mutant Sss1p proteins. Immunoblot analysis was performed on whole cell lysates of yeast cells grown for 8 hours at 30°C as described in (A). Expression of Sec61p and Sbh1p is also shown. (C) Deconvolved widefield microscopy images of yeast strain FKY173 containing an empty plasmid (vector), or expressing wild-type (WT) and Sss1pYG (YG) proteins, and co-transformed with a plasmid expressing the ER marker SRβSA-mRFP (SRβ signal-anchor fused to mRFP. Time points at 3, ,16 and 24 hours of growth in glucose are shown. The cell wall (cyan) was stained blue with Calcofluor White. Scale bar: 7 µm.