Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association.
Plectin is an intermediate filament (IF) binding protein of exceptionally large size. Its molecular structure, revealed by EM and predicted by its sequence, indicates an NH2-terminal globular domain, a long rodlike central domain, and a globular COOH-terminal domain containing six highly homologous repeat regions. To examine the role of the various domains in mediating plectin's interaction with IFs, we have constructed rat cDNAs encoding truncated plectin mutants under the control of the SV-40 promoter. Mutant proteins expressed in mammalian COS and PtK2 cells could be distinguished from endogenous wild type plectin by virtue of a short carboxy-terminal antigenic peptide (P tag). As shown by conventional and confocal immunofluorescence microscopy, the transient expression of plectin mutants containing all six or the last four of the repeat regions of the COOH-terminus, or the COOH-terminus and the rod, associated with IF networks of both the vimentin and the cytokeratin type and eventually caused their collapse into perinuclear aggregates. Similar effects were observed upon expression of a protein encoded by a full length cDNA construct. Microtubules and microfilaments were unaffected. Unexpectedly, mutants containing the rod without any of the COOH-terminal repeats, accumulated almost exclusively within the nuclei of cells. When the rod was extended by the first one and a half of the COOH-terminal repeats, mutant proteins showed a partial cytoplasmic distribution, although association with intermediate filaments was not observed. Nuclear and diffuse cytoplasmic distribution was also observed upon expression of the NH2-terminal domain without rod. These results indicate that sequences located roughly within the last two thirds of the globular COOH-terminus are indispensable for association of plectin with intermediate filaments in living cells.
Wiche
G
G
Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Austria.
Gromov
D
D
Donovan
A
A
Castañón
M J
MJ
Fuchs
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D015870:Gene Expression; D000882:Haplorhini; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism*; D007614:Macropodidae; D008969:Molecular Sequence Data; D051190:Plectin; D014746:Vimentin / Q000378:metabolism
D007381:Intermediate Filament Proteins; D051190:Plectin; D014746:Vimentin