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. 2021 Feb 16;24(3):102196. doi: 10.1016/j.isci.2021.102196

Figure 1.

Figure 1

Endogenous and overexpressed Psd1 is localized to mitochondria

(A) Psd1 detection range was determined for antisera raised in two rabbits 4,077 (green) and 4,078 (blue) using a standard curve of recombinant Psd1. Bleeds number 4 and 5 were analyzed, respectively. The amount of Psd1 detected in WT mitochondria was calculated (n = 3).

(B) Cell extracts derived from pairs of WT and psd1Δ yeast of the indicated backgrounds grown at 30°C in synthetic complete dextrose (SCD) were treated with EndoH as listed and analyzed by immunoblot using the designated anti-Psd1 antisera; Aac2 served as loading control. psd2Δpsd1Δ yeast stably transformed with WT (::WT) or ER-targeted Psd1 (::ER-Psd1) acted as overexpression and glycosylation controls, respectively. Due to its high relative expression, different exposures of ::ER-Psd1 are shown versus all other samples (indicated by frames) which were resolved on the same gel (n = 3).

(C) Following growth in SCD medium to late log phase, fractions were collected from the indicated yeast strains by differential gravity centrifugation. Equal protein amounts from each collected fraction were resolved by SDS-PAGE and immunoblotted for Psd1 and mitochondrial (Qcr6), ER (Sec62), and cytosolic (Hsp70) markers. SM, starting material, P13, pellet of 13,000xg; P21, pellet of 21,000xg; P40, pellet of 40,000xg; and S40, supernatant of 40,000xg (n = 3).

(D) Mitochondria (P13) and ER (P40) fractions were mock or EndoH treated prior to immunoblot analysis (n = 3).

(E) Tandem mass tag (TMT) comparison of ER (P40) proteomes from psd2Δpsd1Δ (n = 3 preps) and psd2Δpsd1Δ::WT yeast (n = 4 preps).

(F) Representative images of HEK293 cells overexpressing FLAG-tagged WT PISD obtained via immunofluorescence to visualize PISD (anti-FLAG; green), mitochondria (anti-TOMM20; red), and ER (anti-calnexin; gray). Bottom panels are a magnification of the white-boxed areas shown in the upper panel. Intensity profile for PISD (green), mitochondria (red), and ER (gray) along the pixels indicated by a solid white line. Scale bars, 5 μm.