Structural and biochemical properties of kinesin heavy chain associated with rat brain mitochondria.
Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria-associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane-associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol-free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria-associated kinesin is discussed.
Jellali
A
A
INSERM, U338 Biologie de la Communication Cellulaire, Strasbourg, France.
Metz-Boutigue
M H
MH
Surgucheva
I
I
Jancsik
V
V
Schwartz
C
C
Filliol
D
D
Gelfand
V I
VI
Rendon
A
A
D000251:Adenosine Triphosphatases / Q000032:analysis; D000255:Adenosine Triphosphate; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D002458:Cell Fractionation; D004072:Digitonin; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments; D051381:Rats; D016415:Sequence Alignment; D017421:Sequence Analysis; D013572:Synaptic Vesicles / Q000737:chemistry
D000596:Amino Acids; D008565:Membrane Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins; D004072:Digitonin