Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

Identification of a Novel Nucleotide-Sensitive Microtubule-Binding Protein in HeLa Cells

Janet E. Rickard and Thomas E. Kreis
The Journal of Cell Biology
Vol. 110, No. 5 (May, 1990), pp. 1623-1633
Stable URL: http://www.jstor.org/stable/1614124
Page Count: 11
  • More info
  • Cite this Item
Identification of a Novel Nucleotide-Sensitive Microtubule-Binding Protein in HeLa Cells
Preview not available

Abstract

A protein of M r 170,000 (170K protein) has been identified in HeLa cells, using an antiserum raised against HeLa nucleotide-sensitive microtubule-binding proteins. Affinity-purified antibodies specific for this 170K polypeptide were used for its characterization. In vitro sedimentation of the 170K protein with taxol microtubules polymerized from HeLa high-speed supernatant is enhanced in the presence of an ATP depleting system, but unaffected by the non-hydrolyzable ATP analogue AMP-PNP. In addition, it can be eluted from taxol microtubules by ATP or GTP, as well as NaCl. Thus it shows microtubule-binding characteristics distinct from those of the previously described classes of nucleotide-sensitive microtubule-binding proteins, the motor proteins kinesin and cytoplasmic dynein, homologues of which are also present in HeLa cells. The 170K protein sediments on sucrose gradients at ∼6S, separate from kinesin (9.5S) and cytoplasmic dynein (20S), further indicating that it is not associated with these motor proteins. Immunofluorescence localization of the 170K protein shows a patchy distribution in interphase HeLa cells, often organized into linear arrays that correlate with microtubules. However, not all microtubules are labeled, and there is a significant accumulation of antigen at the peripheral ends of microtubules. In mitotic cells, 170K labeling is found in the spindle, but there is also dotty labeling in the cytoplasm. After depolymerization of microtubules by nocodazole, the staining pattern is also patchy but not organized in linear arrays, suggesting that the protein may be able to associate with other intracellular structures as well as microtubules. In vinblastine-treated cells, there is strong labeling of tubulin paracrystals, and random microtubules induced in vivo by taxol are also labeled by the antibodies. These immunofluorescence labeling patterns are stable to extraction of cells with Triton X-100 before fixation, further suggesting an association of the protein with cytoplasmic structures. In vivo, therefore, the 170K protein appears to be associated with a subset of microtubules at discrete sites. Its in vitro behavior suggests that it belongs to a novel class of nucleotide-sensitive microtubule-binding proteins.

Page Thumbnails

  • Thumbnail: Page 
1623
    1623
  • Thumbnail: Page 
1624
    1624
  • Thumbnail: Page 
1625
    1625
  • Thumbnail: Page 
1626
    1626
  • Thumbnail: Page 
1627
    1627
  • Thumbnail: Page 
1628
    1628
  • Thumbnail: Page 
1629
    1629
  • Thumbnail: Page 
1630
    1630
  • Thumbnail: Page 
1631
    1631
  • Thumbnail: Page 
1632
    1632
  • Thumbnail: Page 
1633
    1633