Sorting out IF networks: consequences of domain swapping on IF recognition and assembly.
Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins.
McCormick
M B
MB
Howard Hughes Medical Institute, Department of Molecular Genetics, University of Chicago, Illinois 60637.
Coulombe
P A
PA
Fuchs
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006801:Humans; D007382:Intermediate Filaments / Q000502:physiology* / Q000648:ultrastructure; D007633:Keratins / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014746:Vimentin / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure
D014746:Vimentin; D007633:Keratins; D004247:DNA