Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments.
Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain ('coil 1a' and 'coil 2b', respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.
Kouklis
P D
PD
Programme of Cell Biology, European Molecular Biology Laboratory, Heidelberg, FRG.
Traub
P
P
Georgatos
S D
SD
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D016384:Consensus Sequence; D007382:Intermediate Filaments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D011487:Protein Conformation; D014746:Vimentin / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D000911:Antibodies, Monoclonal; D010455:Peptides; D014746:Vimentin