Abstract
MSF, a mitochondrial import stimulation factor purified from rat liver cytosol, is an ATP-dependent precursor protein conformational modulator. As a step toward understanding the specificity of substrate recognition by MSF, various synthetic peptides were examined for their ability to induce MSF ATPase activity. The peptides corresponding to various mitochondria-targeting signal sequences elicited significant ATPase activity. MSF bound the synthetic mitochondrial signal peptides, and ATP hydrolysis caused dissociation of the peptides from MSF. Basic amino acid residues in the signal peptides seemed to be essential for recognition. Thus, MSF is a member of the polypeptide chain-binding protein family with unique recognition specificity and is distinct from the hsp70 family of proteins.
Highlights
EXPERIMENTAL PROCEDURESMost mitochondrial proteins are synthesized on cytoplasmic ATPase Assay-Each of the chemicallysynthesized peptides in 10mM ribosomes and post-translationally imported into mitochondriHa EPESKOH buffer (pH 7.4) was added to the ATPase assay mixture depending on the hydrolysis of cytoplasmic ATP, t h e electro- (20 p1) at concentrations of 0, 0.25, 1, and 2 mM, and the reaction was chemical potential across the inner membrane, and ATtPhein started by the addition of 1pg of MSF (see Fig. 1)or hsp7O (see Fig. 2)
Understanding the specificity of substrate recognition subunits that unfolds the aggregated, import-incompetent prebyMSF, various synthetic peptides were examined for cursors with the dissipation of ATP and binds tothe unfolded their ability to induce MSFATPase activity
We have shown that MSF is a cytoplasmic molecular chaperone that functions in precursor targeting to mitochondria [13, 14].We speculate that MSF maintains the import competence of precursor proteins at theexpense ofATP hydrolysis until the
Summary
Most mitochondrial proteins are synthesized on cytoplasmic ATPase Assay-Each of the chemicallysynthesized peptides in 10mM ribosomes and post-translationally imported into mitochondriHa EPESKOH buffer (pH 7.4) was added to the ATPase assay mixture depending on the hydrolysis of cytoplasmic ATP, t h e electro- (20 p1) at concentrations of 0, 0.25, 1, and 2 mM, and the reaction was chemical potential across the inner membrane, and ATtPhein started by the addition of 1pg of MSF (see Fig. 1)or hsp7O (see Fig. 2). ATP ing 20 mM Tris-HC1 (pH 7.51, 50 mM potassium acetate, 2 mM magnein the matrix is required for the concerted actioonf two mitochondrial heat shock proteins, mhsp7O and hsp; the former binds to the translocating precursors to provide the driving force forpeptidetranslocation, and the latter mediates the sium acetate, and 5 p~ SCC-(1-19) or SCC-(l-lSM) with or without 1 p~ MSF was incubated at 30 "C for 30 min. TheATP-dependentstepoutside of the organelle, on the other hand, has not been extensively characterized.hsp7O has been shown to be involvedin the import of precursor proteins into mitochondria, andhiats been speculatedthat hsp keeps min of incubation,ATP orAMP-PNP was addeadt a final concentration of 1 m ~ a,nd incubation was continued at 30"Cfor min, followed by gel filtration. Another cytosolic factor sensitive to N-ethylmaleimide has been reported to be involved in yeast mitochondrial protein import, and it was speculatedthat this factor functions in precursor recognitioannd mitochondria targeting in yeast [12]
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