Abstract
It has been reported that the expression of TMEM65 is regulated by steroid receptor RNA activator (SRA). To date, however, the localization and function of TMEM65 remained unknown. We analyzed the intracellular localization of TMEM65. Immunoblot and immunostaining analysis revealed mitochondrial localization of TMEM65. Alkali extraction analysis and digitonin extraction test using isolated mitochondria revealed that TMEM65 is an integral membrane protein that localizes to the inner-membrane of mitochondria. Analysis using deletion mutants of TMEM65 suggested that the N-terminal region (1–20) of this protein is sufficient for mitochondrial targeting and that this mitochondrial targeting signal (MTS) is cleaved between the amino acid positions 35 and 64, which contain a putative recognition site of matrix processing protease (MPP). Together, these results suggest that TMEM65 is imported into the mitochondria, integrated into mitochondrial inner-membrane, and processed into its mature form by an MPP.
Highlights
The mitochondrion is an organelle compartmented by two membranes (Pfanner & Wiedemann, 2002), which divide this organelle into four compartments
Some of these proteins are synthesized in the cytosol as precursors harboring mitochondrial targeting signals (MTS) at their N-terminus, which is imported into the mitochondria and processed into mature form by a matrix processing protease (MPP) that cleaves and removes the MTS (Pfanner & Wiedemann, 2002; Gakh, Cavadini & Isaya, 2002)
Results of the PSPORT prediction analysis suggested that human TMEM65 had three putative transmembrane regions (Fig. 1A) and a putative recognition site (RRL | GT between the amino acid residues 52–56 of hTMEM65) of mitochondrial MPP (R-2 motif, XRX | X(S/X)) (Gakh, Cavadini & Isaya, 2002)
Summary
The mitochondrion is an organelle compartmented by two membranes (outer-membrane and inner-membrane) (Pfanner & Wiedemann, 2002), which divide this organelle into four compartments (outer-membrane, intermembrane space, inner-membrane and matrix). Most mitochondrial proteins are coded in the genomic DNA, synthesized in cytosol, and imported into mitochondria (Koopman et al, 2013). Some of these proteins are synthesized in the cytosol as precursors harboring mitochondrial targeting signals (MTS) at their N-terminus, which is imported into the mitochondria and processed into mature form by a matrix processing protease (MPP) that cleaves and removes the MTS (Pfanner & Wiedemann, 2002; Gakh, Cavadini & Isaya, 2002). How to cite this article Nishimura et al (2014), TMEM65 is a mitochondrial inner-membrane protein.
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