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Structural and biochemical properties of kinesin heavy chain associated with rat brain mitochondria
A Jellali1, M H Metz-Boutigue, I Surgucheva
1INSERM, U338 Biologie de la Communication Cellulaire, Strasbourg, France.
Abstract:
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.
Insights
Kinesin, a motor protein, is found associated with mitochondria in rat brains. This protein, conventional kinesin, may play a role in mitochondrial function and exists in various cellular forms.
Area of Science:
- Molecular Biology
- Cell Biology
- Neuroscience
Background:
- Kinesin is a motor protein crucial for intracellular transport of membranous organelles.
- While primarily known from soluble brain extracts, kinesin's association with intracellular organelles is suggested by prior studies.
Purpose of the Study:
- To investigate the presence and characteristics of kinesin associated with intracellular membranous organelles in the rat brain.
- To determine the localization and properties of kinesin within mitochondria.
Main Methods:
- Immunocytochemistry using an antibody against Drosophila kinesin heavy chain.
- Differential centrifugation and immunoblotting to detect kinesin in rat brain microsomes, synaptic vesicles, and mitochondria.
- Low salt and ATP extraction from mitochondria.
- Limited sequence analysis of the solubilized protein.
- Digitonin fractionation and KI extraction of mitochondria.
- Comparison of soluble and membrane-associated kinesin isoforms using polyclonal antibodies.
Main Results:
- A 116 kDa protein, identified as conventional kinesin, was detected in rat brain microsomes, synaptic vesicles, and mitochondria.
- This mitochondrial kinesin could be extracted with low salt or ATP, indicating a peripheral association.
- Kinesin is localized to a cholesterol-free domain in the outer mitochondrial membrane, potentially at contact sites with the inner membrane.
- Soluble and mitochondrial-associated kinesins exhibit different isoform patterns, suggesting differential cellular distribution.
Conclusions:
- Kinesin is associated with mitochondria in the rat brain, specifically on the outer mitochondrial membrane.
- The distinct isoform patterns suggest that kinesin exists as multiple isoforms with specialized cellular roles.
- Mitochondria-associated kinesin may be functionally regulated and its localization at membrane contact sites could be significant.