Characterization of interactions between the neurofilament triplet proteins by the yeast two-hybrid system.
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 alpha-helical rod region of approximately310 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 alpha-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.
Leung
C L
CL
Department of Biochemistry, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Liem
R K
RK
D000818:Animals; D001369:Axons / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids; D055503:Protein Multimerization; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D014162:Transfection; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000096:biosynthesis
D016900:Neurofilament Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase