STRUCTURAL AND BIOCHEMICAL-PROPERTIES OF KINESIN HEAVY-CHAIN ASSOCIATED WITH RAT-BRAIN MITOCHONDRIA

Kinesin, a mechanochemical enzyme that translocates membranous organel les, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological appro aches have demonstrated that kinesin could be associated to intracellu lar 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 diff erential centrifugation and immunoblotting we observed a 116 kDa prote in that crossreacts with this antibody in microsomes, synaptic vesicle s, 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 mitoc hondria-associated kinesin recognizes soluble bovine brain kinesin. Th e soluble and mitochondrial membrane-associated kinesins show a differ ent isoform pattern. These results are consistent with the idea that k inesin exists as multiple isoforms that might be differentially distri buted within the cell. In addition digitonin fractionation of mitochon dria 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 mit ochondrial outer and inner membranes. The significance of these observ ations on the functional regulation of the mitochondria-associated kin esin is discussed. (C) 1994 Wiley-Liss, Inc.