A High Molecular Weight Intermediate Filament-associated Protein in BHK-21 Cells Is Nestin, a Type VI Intermediate Filament Protein

doi:10.1074/jbc.274.14.9881

A High Molecular Weight Intermediate Filament-associated Protein in BHK-21 Cells Is Nestin, a Type VI Intermediate Filament Protein

LIMITED CO-ASSEMBLY IN VITRO TO FORM HETEROPOLYMERS WITH TYPE III VIMENTIN AND TYPE IV α-INTERNEXIN*

From the ^‡Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752,^¶Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3072, and ^‖Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand

Abstract

BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein α-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF110498

The amino acid sequence of this protein can be accessed through the Swiss Protein Database (Siss-Prot P21263).
↵§ To whom correspondence should be addressed: National Institutes of Health, Bldg, 6, Rm. 425, 9000 Rockville Pike, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail:pemast{at}helix.nih.gov.
↵** Supported by National Institutes of Health Grant GM36806-4.
Abbreviations:

IF

intermediate filament(s)

BHK-21

baby hamster kidney cells, clone 21

DTSSP

3,3′-dithiobis(sulfosuccinimidyl propionate)

IFAP

intermediate filament-associated protein(s)

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

FPLC

fast protein liquid chromatography

PCR

polymerase chain reaction

nt

nucleotide

bp

base pair
- Received December 8, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.