Cell Biology

Published February 10, 2003

JCB Article
Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane
Ryen D. Fons, Brigitte A. Bogert, and Ramanujan S. Hegde
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

The Journal of Cell Biologylthough the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the transloconassociated protein(TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not

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due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property ofthe signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as afunctional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.

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Introduction
The vast majority of secretory and membrane proteins are transported across or integrated into the membrane of the mammalian ER by a multiprotein assembly termed the translocon (forreview see Johnson and van Waes, 1999). An essential function of the translocon is to provide an aqueous protein-conducting channel spanning the membrane bilayer. At the ER, the heterotrimeric Sec61p complex (composed of , , and subunits) has been shown to be a principal component of this translocation channel (Gorlich et al., 1992b; Musch et al., 1992; Sanders et al., 1992; Mothes et al., 1994).This protein complex can homo-oligomerize into a toroidal channel-like structure (Hanein et al., 1996; Beckmann et al., 1997), bind ribosomes with high affinity (Gorlich et al., 1992b; Kalies et al., 1994), recognize functional signal sequences (Jungnickel and Rapoport, 1995), and allow the lateral partitioning of transmembrane domains into the lipid bilayer (Heinrich et al., 2000).Notwithstanding this multifunctionality, the Sec61p complex is insufficient for cotranslational translocation. Although the Sec61p complex can recognize signal sequences, efficient nascent chain targeting to the membrane and transfer to the translocation channel additionally requires the coordinated actions of the cytosolic signal recognition particle and its membrane-bound receptor (SR)* (Walter and Johnson,1994). After targeting to the mammalian ER, another component, TRAM, is necessary for the efficient translocation of most substrates in a manner dependent on structural features of their signal sequences (Gorlich et al., 1992a; Voigt et al., 1996). Thus, reconstitution into lipid vesicles of at least three proteins, the Sec61p complex, SR, and TRAM, is necessary for the translocation of mostsubstrates, defining them as essential translocon components (Gorlich and Rapoport, 1993).
*Abbreviations used in this paper: ConA, concanavalin A; CNX, calnexin; DBC, DeoxyBigCHAP; EB, extraction buffer; GH, growth hormone; Ost, osteopontin; PK, proteinase K; Prl, prolactin; PrP, prion protein; RAMP, ribosome-associated membrane protein; RM, rough microsomal membrane; SR, signal recognition...