Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin-containing cells.
Using immunoelectron microscopy it is demonstrated that desmin subunits missing their complete carboxy-terminal domain are incapable of homopolymeric filament formation in vivo. Furthermore it is shown that, in vimentin-containing cells, desmin integrates into preexisting vimentin filaments resulting in desmin/vimentin heteropolymers. Removal of the amino-terminal or both nonhelical end domains of desmin increases Triton X-100 solubility of the mutant desmin subunits. Expression of desmin mutants containing deletions in the C-terminal part of the rod in vimentin-free cells results in an increase of the Triton X-100 solubility too. In contrast, if expressed in vimentin-containing cells, these mutant subunits remain in the Triton X-100 insoluble fraction. Deletion of the nonhelical carboxy-terminal domain only has no effect on solubility. In vimentin-free cells, stably expressed desmin subunits missing their amino-terminal domains display a slightly higher turnover rate compared to wild-type desmin. Transiently expressed desmin subunits missing 18 or more carboxy-terminal residues of the rod domain are rapidly degraded in vimentin-free cells. In vimentin-containing cells, turnover rates were much less pronounced. Finally, by using site-directed mutagenesis, we were able to map specific residues important for de novo filament assembly within the amino-terminal domain and in the conserved part at the C-terminus of the alpha-helical domain.
Raats
J M
JM
Department of Biochemistry, University of Nijmegen, The Netherlands.
Gerards
W L
WL
Schreuder
M I
MI
Grund
C
C
Henderik
J B
JB
Hendriks
I L
IL
Ramaekers
F C
FC
Bloemendal
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D003893:Desmin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry / Q000648:ultrastructure*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014746:Vimentin / Q000378:metabolism*
D003893:Desmin; D014746:Vimentin