Neurofilament Protein Heterotetramers as Assembly Intermediates

doi:10.1074/jbc.270.16.9334

Neurofilament Protein Heterotetramers as Assembly Intermediates (*)

From the (1) Department of Chemistry and Biochemistry, California State University, Long Beach, California 90840

^§ To whom correspondence should be addressed:
Dept. of Chemistry and Biochemistry, California State University, Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840.
Tel.: 310-985-4944; Fax: 310-985-2315.

Abstract

Evidence is presented for the existence of a soluble heterotetramer containing the low and middle molecular weight neurofilament (NF) proteins, NF-L and NF-M, and one containing the low and high molecular weight proteins, NF-L and NF-H, and for their role in filament assembly. When a mixture of either pair of proteins was renatured in 2 M urea, 20 m M Tris, pH 7.2, a new band representing a complex was observed in native gel electrophoresis. No new band was observed with a mixture of NF-M and NF-H. Two-dimensional gel electrophoresis showed that treatment of the complexes with SDS caused them to dissociate into their constituent polypeptide chains. Native neurofilaments dissociated in 2 M urea into a mixture of LM and LH complexes. Titration of NF-L with NF-M indicated that complex formation was complete at an approximately equimolar ratio of the two proteins. The LM complex had a sedimentation coefficient, s, of 4.4 S, consistent with a tetrameric structure. Dialysis of a solution of the LM complex against 50 m M 4-morpholineethanesulfonic acid, 0.17 M NaCl, pH 6.25, led to the formation of 10-nm filaments in good yield. These results suggest that NF protein heterooligomers are intermediates in NF assembly and disassembly.

Footnotes

↵* This work was supported by National Science Foundation Grants DCB-8902594 and DCB-8904460, by a summer fellowship (to T. Tran) from the Howard Hughes Medical Institute Biological Sciences Initiative, and by the California State University, Long Beach, CA. The purchase of the electron microscope was funded in part by National Science Foundation Grant BBS 88-20774. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NF

neurofilament

NF-L

NF-M and NF-H, low, middle, and high molecular weight neurofilament proteins

DTT

dithiothreitol

IF

intermediate filament

PMSF

phenylmethylsulfonyl fluoride

PIPES

1,4-piperazinediethanesulfonic acid

TEMED

N,N,N′, N′-tetramethylethylenediamine

MES

4-morpholineethanesulfonic acid.
↵²T. Tran, A. Ansari, J. Cohlberg, M. Lee, and D. Cleveland, unpublished experiments.