RESUMO
Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.
Assuntos
Retículo Endoplasmático , Dobramento de Proteína , Humanos , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/química , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/química , Células HEK293 , Domínios Proteicos , Interações Hidrofóbicas e Hidrofílicas , Processamento de Proteína Pós-Traducional , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/químicaRESUMO
Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the 'positive-inside' rule.