Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.
We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.
Nikolic
B
B
Vienna Biocenter, Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Austria.
Mac Nulty
E
E
Mir
B
B
Wiche
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D018407:Bacterial Physiological Phenomena; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000502:physiology; D003599:Cytoskeleton / Q000737:chemistry; D015870:Gene Expression / Q000502:physiology; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D015603:Keratinocytes / Q000502:physiology; D007633:Keratins / Q000378:metabolism; D007614:Macropodidae; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051190:Plectin; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014746:Vimentin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D000596:Amino Acids; D007381:Intermediate Filament Proteins; C495333:Plec protein, mouse; D051190:Plectin; D011994:Recombinant Proteins; D014746:Vimentin; D007633:Keratins