Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M.
We have generated a set of amino- and carboxy-terminal deletions of the neurofilament NF-M gene and determined the molecular consequences of forced expression of these mutant constructs in mouse fibroblasts. To follow the expression of mutant NF-M subunits in transfected cells, a 12 amino acid epitope (from the human c-myc protein) was expressed at the carboxy terminus of each mutant. We show that NF-M molecules missing up to 90 or 70% of the nonhelical carboxy-terminal tail or amino-terminal head domains, respectively, incorporate readily into an intermediate filament network comprised either of vimentin or NF-L, whereas deletions into either the amino- or carboxy-terminal alpha-helical rod region generate assembly-incompetent polypeptides. Carboxy-terminal deletions into the rod domain invariably yield dominant mutants which rapidly disrupt the array of filaments comprised of NF-L or vimentin. Accumulation of these mutant NF-M subunits disrupts vimentin filament arrays even when present at approximately 1% the level of the wild-type subunits. In contrast, the amino-terminal deletions into the rod produce pseudo-recessive mutants that perturb the wild-type NF-L or vimentin arrays only modestly. The inability of such amino-terminal mutants to disrupt wild-type subunits defines a region near the amino-terminal alpha-helical rod domain (residues 75-126) that is required for the earliest steps in filament assembly.
Wong
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PC
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Cleveland
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DW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007382:Intermediate Filaments / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D014746:Vimentin / Q000502:physiology
D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D014746:Vimentin