RESUMO
Isoforms of importin-α have been identified in insect and human cells, and cross-linking experiments suggest that at least one isoform in each species participates in the targeting of integral membrane proteins to the inner nuclear membrane (INM). To directly test this hypothesis, an assay was developed using Saccharomyces cerevisiae. The data show that internal promoters are present within KAP60, and the nested transcripts are translated into three isoforms: Kap60-44, Kap60-30 and Kap60-10. In the absence of the isoforms, the INM protein Heh2-green fluorescent protein (GFP) localized to cytoplasmic membranes, whereas its wild-type localization at the nuclear periphery was restored when the Kap60-44 isoform was reintroduced. An INM-sorting sequence has been identified that cross-links with the isoform of importin-α that directs trafficking toward the nuclear envelope (NE). When this sequence in HEH2 was mutated, Heh2 was again localized to cytoplasmic membranes. Thus, this report provides the first evidence that isoforms of Kap60 exist in yeast, and these isoforms participate in the molecular sorting and enrichment of INM proteins to the NE. Herein, we provide additional support for the hypothesis that trafficking of INM proteins to the NE is a regulated process.
Assuntos
Carioferinas/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/metabolismo , Sequência de Aminoácidos , Carioferinas/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Nucleares , Regiões Promotoras Genéticas , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , alfa Carioferinas/genéticaRESUMO
Spodoptera frugiperda (Sf9) importin-alpha-16 is a translocon-associated protein that participates in the early sorting pathway of baculovirus integral membrane proteins destined for the inner nuclear membrane (INM). To discern whether sorting intermediate protein complexes like those observed in insect cells are also formed with mammalian INM proteins, cross-linked complexes of importin-alpha-16 with human lamin B receptor (LBR) and nurim were examined. Both LBR and nurim cross-link with Sf9 importin-alpha-16 during cotranslational membrane integration and remain proximal with importin-alpha-16 after integration into the endoplasmic reticulum membrane and release from the translocon. Human cells encode several isoforms of importin-alpha; to determine whether any of these isoforms may recognize INM-directed proteins, they were tested for their ability to cross-link with the viral-derived INM sorting motif sequence. One cross-linked adduct was detected with a 16-kDa isoform encoded from KPNA4 (KPNA-4-16). KPNA-4-16 was easily detected in microsomal membranes prepared from KPNA4-16 recombinant virus-infected cells and was also detected in microsomes prepared from HeLa cells. Together these observations suggest that elements of the early sorting pathway of INM-directed proteins mediated by importin-alpha-16 are highly conserved, and mammalian KPNA-4-16 is a candidate partner in sorting integral membrane proteins to the INM.