Characterization of Interactions between the Neurofilament Triplet Proteins by the Yeast Two-hybrid System

doi:10.1074/jbc.271.24.14041

Characterization of Interactions between the Neurofilament Triplet Proteins by the Yeast Two-hybrid System*

From the Departments of^‡ Biochemistry and Molecular Biophysics and
^¶ Pathology and
^∥ Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032

^” To whom correspondence should be addressed:
Dept. of Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032.
Tel.: 212-305-4078; Fax: 212-305-5498. E-mail: RKL2{at}columbia.edu

Abstract

In the adult axon, the neurofilaments (NFs) are heteropolymers formed from the low (NFL), middle (NFM), and high (NFH) molecular weight neurofilament triplet proteins (NFTPs). All three proteins have the basic intermediate filament protein tripartite structure, which consists of a short amino-terminal head region, an α-helical rod region of ∼310 amino acids, and a carboxyl-terminal tail region of variable length. In vitro polymerization studies have shown that only NFL can assemble into homopolymeric 10-nm filaments. The assembly of intermediate filaments, including the NFs, begins with the formation of a coiled-coil dimer involving the α-helical rod domains of two molecules. In order to determine whether homodimers or heterodimers of NFTPs are the preferred intermediates in the assembly of NFs, we have used the yeast two-hybrid system to study the interactions between the different NFTPs. By monitoring the activity of the lacZ reporter gene product, we are able to show that the interactions of NFL with NFL, NFM, or NFH are stronger than the interactions of NFM with NFM or NFH and the interaction of NFH with NFH. These results imply that NFM and NFH are more likely to form heterodimers with NFL than homodimers and are consistent with the inability of NFM and NFH to self-polymerize in vitro and in vivo.

Footnotes

↵^§ Supported by Training Grants EY07105 and AG00189 as a National Institutes of Health predoctoral trainee.
↵* This work was supported by National Institutes of Health Grant NS15182 (to R. K. H. L.). 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.
↵¹ The abbreviations used are:

NF

neurofilament

IF

intermediate filament

NFH

NFM, and NFL, high, middle, and low molecular weight neurofilament subunits

HΔ

NFH^1-415

MΔ

NFM^1-421

LΔ

NFL^1-415

ΔMΔ

NFM^44-421

ΔLΔ

NFL^24-415

kb

kilobase(s)

NFTP

neurofilament triplet proteins.
- Received December 20, 1995.
- Revision received March 6, 1996.