ASSOCIATION OF KINESIN WITH THE GOLGI-APPARATUS IN RAT HEPATOCYTES

The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle an d after microtubule disruption. These dynamics are believed to be supp orted by a close structural interaction of the Golgi with the microtub ule cytoskeleton and associated motor enzymes. One microtubule-depende nt motor enzyme, kinesin, has been implicated in Golgi movement and fu nction although direct evidence supporting this interaction is lacking . In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous org anelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents similar to 0.3% of total protein in rat liver homogenates, with similar to 30% membrane-associated and the re mainder in the cytosol. Among membrane fractions, kinesin was concentr ated markedly in Golgi-enriched fractions, which were prepared using t wo independent techniques. Kinesin was also abundant in fractions enri ched in transcytotic carriers and secretory vesicles, with lower level s detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mitochondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultu red hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes wit h the Golgi marker, alpha-mannosidase II, in both cell types. These re sults provide compelling evidence showing that kinesin is associated w ith the Golgi complex in cells and implicate this motor enzyme in Golg i structure, function and dynamics.