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. 2017 Sep 19;7(11):3731–3743. doi: 10.1534/g3.117.300227

Figure 3.

Figure 3

Loss of Psh1p or its ubiquitin ligase activity affects the steady-state levels of many proteins when expressed from plasmids, but not when expressed from the chromosome. (A, B) The steady-state protein levels of CEN-plasmid expressed mia40-4pHA (A) or Fzo1pHA (B) were analyzed in psh1Δ cells coexpressing either vector, WT PSH1HA, or RING domain mutant PSH1HAC45S C50S growing at 37° (mia40-4pHA) or 30° (Fzo1pHA) by immunoblotting with HA antibody. Anti-PGK serves as a loading control. (C) Levels of the mitochondrial proteins Mia40pHA, erv1-2pHA, and mitochondrial-targeted GFP (mtGFP) expressed from CEN plasmids in WT and psh1Δ cells were assessed in WT and psh1Δ cells at 30° as in (A). (D) CEN plasmid-expressed Ura3pHA-CL1, Ste6p*HA, and CPY*HA were analyzed in WT and psh1Δ cells at 30° as in (A) except CPY*HA was visualized using anti-CPY. (E) Chromosomal proteins (CPY, PGK, and Porin) were analyzed in WT and psh1Δ cells by immunoblotting using antibodies specific to these targets. (F, G). Sam35pHA (F) or Sen2pHA (G) expressed from either the genome or a CEN plasmid were analyzed in WT and psh1Δ cells at 30° as in (A). (H–J) CEN plasmid-expressed Fzo1pHA under control of the ADH1 promoter (H) or mia40-4pHA and Mia40pHA under control of the GAL10 promoter (I) or with the native MIA40 3′ untranslated region (J) were assessed in psh1Δ and WT cells at 30° (Fzo1pHA) or 37° (mia40-4pHA), as in (A).