Identification of a keratin-associated protein that localizes to a membrane compartment.
We describe the characterization of an acidic glycoprotein (molecular mass approximately 85 kDa) that associates with keratin intermediate filaments of 'simple'-type epithelia. Using a number of anti-keratin monoclonal antibodies, the 85 kDa glycoprotein was identified by co-immunoprecipitation with keratin polypeptides 8 and 18 (K8/18) from the human colonic epithelial cell line HT29 and several other epithelial cell lines. This Keratin-Associated Protein (termed KAP85) was readily detected after in vitro galactosylation of K8/18 immunoprecipitates obtained from mitosis-arrested cells. Its solubilization and detection were dependent on the detergent used, and it was barely detected after in vitro galactosylation of asynchronously growing G0/G1-phase cells. Its poor in vitro galactosylation in G0/G1-phase cells is likely a reflection of the lack of available terminal N-acetylglucosamine residues, since it can be labelled to a similar extent in G0/G1- and G2/M-phase cells using NaIO4/NaB3H4. Glycosidase digestion showed that KAP85 contains high mannose and complex oligosaccharides. Fractionation of total cellular K8/18 into soluble and cytoskeletal insoluble pools showed that KAP85 associates exclusively with the cytoskeletal K8/18 pool. Subcellular fractionation showed that KAP85 co-localizes with a plasma-membrane-enriched fraction that includes the transferrin receptor and KS-1 antigen. Our results demonstrate in vitro evidence of a membrane-associated glycoprotein (KAP85) which may serve as an attachment site for filamentous K8/18.
Chou
C F
CF
Palo Alto Veterans Administration Medical Center, CA 94304.
Riopel
C L
CL
Omary
M B
MB
D002451:Cell Compartmentation; D002453:Cell Cycle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007633:Keratins / Q000737:chemistry / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D011506:Proteins; D007633:Keratins; D008358:Mannose