The Sec61p Complex Is a Dynamic Precursor Activated Channel

Abstract

Previous studies have shown that the rough endoplasmic reticulum (ER) contains nascent precursor polypeptide gated channels. Circumstantial evidence suggests that these channels are formed by the Sec61p complex. We reconstituted the purified Sec61p complex in a lipid bilayer and characterized its dynamics and regulation. The Sec61p complex is sufficient to form the precursor polypeptide activated channel under co- and posttranslational transport conditions. Activity of the Sec61p channel in both transport modes is induced by direct interaction with precursor protein. The Sec61p complex comprises a highly dynamic pore covering conductances corresponding to channel openings from approximately 6 to 60 A. Its properties are indistinguishable from those we observed with native ER channels, directly demonstrating that these channels are formed by the Sec61p complex.

Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum

“…Single Channel Recordings-Vesicles for planar bilayer experiments were prepared by mixing (3:2, v/v) the Sec61-containing vesicles with preformed liposomes (egg L-␣-phosphatidylcholine, 10 mg/ml) in 50 mM KCl and 10 mM Mops/Tris (pH 7.0) (9). Mega-9 (nonanoyl-N-methylglucamide) was added to a final concentration of 80 mM.…”

“…Membrane potentials refer to the trans compartment. Data recording and analysis were performed as described previously (9). Voltage ramps were conducted at a rate of 6.6 mV/s.…”

“…The protein biogenesis function involves an aqueous polypeptide-conducting pore in the ER membrane, which is formed by the heterotrimeric Sec61 complex (5)(6)(7)(8)(9)(10)(11); the storeand receptor-controlled Ca 2ϩ release function requires a steep ER to cytosol gradient, with 500 -800 M free Ca 2ϩ in the ER. Recent work demonstrated that the Sec61 complex can transiently allow passive ER Ca 2ϩ efflux (11)(12)(13)(14).…”

“…In addition, the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) counteracts Ca 2ϩ leakage from the ER (15). The Hsp70-type molecular chaperone BiP plays a role in Sec61 channel closure (8,9,16,17) (Fig. 1).…”

“…Furthermore, it was observed in vitro as well as in intact cells that the ER lumenal Hsp70-type molecular chaperone, BiP, is involved in limiting ER Ca 2ϩ leakage at the level of the Sec61 complex (8,9,16,17,53,60) (Fig. 1).…”