The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

• MeSH
• Biotechnology methods   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Kinesins * chemistry   genetics   immunology   metabolism   MeSH
• Cloning, Molecular MeSH
• Humans MeSH
• Epitope Mapping * MeSH
• Antibodies, Monoclonal immunology   MeSH
• Plasmids MeSH
• Recombinant Proteins * chemistry   genetics   immunology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Kinesins * MeSH
• Antibodies, Monoclonal MeSH
• Recombinant Proteins * MeSH

gamma-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that gamma-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of gamma-tubulin with alphabeta-tubulin dimers. gamma-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large gamma-tubulin complexes resistant to salt treatment were found to be associated with a high-speed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was >1 MD. Large gamma-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate gamma-tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of gamma-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of gamma-tubulin suggests additional roles for this protein species in microtubule organization.

• MeSH
• Antibodies, Antinuclear genetics   metabolism   MeSH
• Arabidopsis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cytosol metabolism   MeSH
• Dimerization MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fluorescent Antibody Technique MeSH
• Microtubules metabolism   MeSH
• Microsomes metabolism   MeSH
• Mitosis physiology   MeSH
• Precipitin Tests MeSH
• Arabidopsis Proteins metabolism   MeSH
• Tubulin chemistry   immunology   metabolism   MeSH
• Protein Binding MeSH
• Vicia faba metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antibodies, Antinuclear MeSH
• Arabidopsis Proteins MeSH
• Tubulin MeSH