RESUMO
TorsinA (also known as torsin-1A) is a membrane-embedded AAA+ ATPase that has an important role in the nuclear envelope lumen. However, most torsinA is localized in the peripheral endoplasmic reticulum (ER) lumen where it has a slow mobility that is incompatible with free equilibration between ER subdomains. We now find that nuclear-envelope-localized torsinA is present on the inner nuclear membrane (INM) and ask how torsinA reaches this subdomain. The ER system contains two transmembrane proteins, LAP1 and LULL1 (also known as TOR1AIP1 and TOR1AIP2, respectively), that reversibly co-assemble with and activate torsinA. Whereas LAP1 localizes on the INM, we show that LULL1 is in the peripheral ER and does not enter the INM. Paradoxically, interaction between torsinA and LULL1 in the ER targets torsinA to the INM. Native gel electrophoresis reveals torsinA oligomeric complexes that are destabilized by LULL1. Mutations in torsinA or LULL1 that inhibit ATPase activity reduce the access of torsinA to the INM. Furthermore, although LULL1 binds torsinA in the ER lumen, its effect on torsinA localization requires cytosolic-domain-mediated oligomerization. These data suggest that LULL1 oligomerizes to engage and transiently disassemble torsinA oligomers, and is thereby positioned to transduce cytoplasmic signals to the INM through torsinA.
Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Membrana Nuclear/metabolismo , Células 3T3 , Adenosina Trifosfatases/metabolismo , Animais , Células CHO , Proteínas de Transporte/genética , Linhagem Celular , Cricetulus , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
The nuclear pore complex (NPC) plays a critical role in gene expression by mediating import of transcription regulators into the nucleus and export of RNA transcripts to the cytoplasm. Emerging evidence suggests that in addition to mediating transport, a subset of nucleoporins (Nups) engage in transcriptional activation and elongation at genomic loci that are not associated with NPCs. The underlying mechanism and regulation of Nup mobility on and off nuclear pores remain unclear. Here we show that Nup50 is a mobile Nup with a pronounced presence both at the NPC and in the nucleoplasm that can move between these different localizations. Strikingly, the dynamic behavior of Nup50 in both locations is dependent on active transcription by RNA polymerase II and requires the N-terminal half of the protein, which contains importin α- and Nup153-binding domains. However, Nup50 dynamics are independent of importin α, Nup153, and Nup98, even though the latter two proteins also exhibit transcription-dependent mobility. Of interest, depletion of Nup50 from C2C12 myoblasts does not affect cell proliferation but inhibits differentiation into myotubes. Taken together, our results suggest a transport-independent role for Nup50 in chromatin biology that occurs away from the NPC.
Assuntos
Diferenciação Celular , Cromatina/genética , Citoplasma/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Animais , Técnicas de Cultura de Células , Regulação da Expressão Gênica , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , RNA/genética , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMO
The RGS7 (R7) family of G protein regulators, Gbeta5, and R7BP form heterotrimeric complexes that potently regulate the kinetics of G protein-coupled receptor signaling. Reversible palmitoylation of R7BP regulates plasma membrane/nuclear shuttling of R7*Gbeta5*R7BP heterotrimers. Here we have investigated mechanisms whereby R7BP controls the function of the R7 family. We show that unpalmitoylated R7BP undergoes nuclear/cytoplasmic shuttling and that a C-terminal polybasic motif proximal to the palmitoylation acceptor sites of R7BP mediates nuclear localization, palmitoylation, and plasma membrane targeting. These results suggest a novel mechanism whereby palmitoyltransferases and nuclear import receptors both utilize the C-terminal domain of R7BP to determine the trafficking fate of R7*Gbeta5*R7BP heterotrimers. Analogous mechanisms may regulate other signaling proteins whose distribution between the plasma membrane and nucleus is controlled by palmitoylation. Lastly, we show that cytoplasmic RGS7*Gbeta5*R7BP heterotrimers and RGS7*Gbeta5 heterodimers are equivalently inefficient regulators of G protein-coupled receptor signaling relative to plasma membrane-bound heterotrimers bearing palmitoylated R7BP. Therefore, R7BP augments the function of the complex by a palmitoylation-regulated plasma membrane-targeting mechanism.