Article

cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol.

Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Journal of Biochemistry (impact factor: 2.37). 09/1994; 116(2):416-25. pp.416-25
Source: PubMed

ABSTRACT We identified a liver cytosolic protein factor that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria. It was termed mitochondrial import stimulation factor or MSF [Hachiya, N. et al. (1993) EMBO J. 12, 1579-1586]. It consisted of 32-kDa (MSFL) and 30-kDa (MSFS) polypeptides as assessed by SDS-PAGE. MSF recognized the presequence portion of mitochondrial precursor proteins and catalyzed the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner. We report here the cDNA cloning and characterization of MSF. Microsequencing of MSFL and MSFS showed that they belonged to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. We cloned the cDNA of MSFL and that of one component of MSFS (MSFS1) from a rat liver cDNA library. The cloned cDNAs were separately expressed in Escherichia coli and the expressed proteins were purified to homogeneity. The purified recombinant MSFL and MSFS1 stimulated mitochondrial import of adrenodoxin precursor (pAd) synthesized in vitro with wheat germ lysate translation system. Recombinant MSFL or MSFS1 had the ability to bind with denatured pAd and they kept the precursor in an import-competent state. Rabbit polyclonal antibodies raised against the recombinant proteins inhibited the import-stimulation activity of rat liver cytosol as well as that of authentic purified MSF. Identification of MSF as 14-3-3 proteins establishes a novel function for this family of proteins and indicates their role as cytosolic chaperones to aid many important cellular events.

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Keywords

adrenodoxin precursor
 
ATP-dependent manner
 
authentic purified MSF
 
cDNA cloning
 
cellular events
 
cloned cDNAs
 
cytosolic chaperones
 
expressed proteins
 
import-competent state
 
import-stimulation activity
 
liver cytosolic protein factor
 
mitochondrial import
 
mitochondrial precursor proteins
 
purified recombinant MSFL
 
Rabbit polyclonal antibodies
 
rat liver cytosol
 
Recombinant MSFL
 
recombinant proteins inhibited
 
vitro synthesized mitochondrial precursor proteins
 
wheat germ lysate-synthesized precursor proteins
 

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