Figure 1.

GRSF1 Is a Mitochondrial Protein

(A) Schematic representation of GRSF1 isoform 1 and 2. MTS, mitochondrial targeting signal predicted by the MitoProt algorithm (http://ihg.gsf.de/ihg/mitoprot.html). qRRM, quasi-RNA-recognition motif. RNAi, targeting sites of GRSF1 RNAi1 and RNAi2. MitoProt, probability of mitochondrial import according to MitoProt algorithm.

(B) Confocal analysis of GRSF1 isoforms. HeLa cells were transfected with GRSF1-isoform1-mCherry (top panel) or GRSF1-isoform2-mCherry (middle panel) and immunolabeled with anti-Tom20. Bottom panel, endogenous GRSF1 and Tom20 were immunolabeled in 143B cells. Arrowheads, foci formed by GRSF1 in mitochondria.

(C) Import of radiolabeled in vitro-translated GRSF1 isoforms into isolated 143B mitochondria. p, precursor. m, mature. ΔΨm, mitochondrial membrane potential. PK, Proteinase K. Note that precursor GRSF1 migrates at ∼70 kDa instead of the predicted 53 kDa, and mature GRSF1 runs at ∼55 kDa instead of the predicted 43 kDa.

(D) Immunoblot analysis of mitochondrial extract after Proteinase K accessibility test. PK, Proteinase K. TX-100, Triton X-100. OPA1 is a mitochondrial intermembrane space protein anchored to the inner membrane; Tom20 is a mitochondrial outer-membrane protein; mtSSB is a mitochondrial matrix protein. Note that mature endogenous GRSF1 migrates at ca 55kDa, similar to in vitro-translated GRSF1 after import (C).

(E) Immunoblot analysis of mitochondrial extract after alkaline sodium carbonate (Na₂CO₃) extraction. T, total extract. S, supernatant. P, pellet. PHB, prohibitin 1. Cyt c, cytochrome c.