Reconstimtion of Protein Translocation from Solubilized Yeast Membranes Reveals Topologically Distinct Roles for BiP and Cytosolic Hsc70

We reconstituted prepro-ot-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc)70 homologs, yeast eytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required eytosolic hse70. We conclude that cytosolic hse'/0 and BiP serve noninterehangeable roles in polypeptide transloeation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane. T HE precise mechanism whereby a charged, polar secretory polypeptide traverses the hydrophobic environment of the ER lipid bilayer remains poorly understood. It is now generally believed that proteins exist to facilitate this process. To identify some of these factors, our group (Deshaies and Schekman, 1987; Rothblatt et al., 1989; Stirring et al., 1992) and others (Toyn et al., 1988; Sadler et al., 1989; Green et al., 1992) have used the yeast Saccharomyces cerevisiae to screen for mutants that accumulate preproteins in the cytosol. Three of these temperature-sensitive mutants were designated sec61, sec62, and sec63. The products of these genes have been identified as integral membrane proteins of the ER (Deshaies and Schekman, 1987; Deshaies and Schekman, 1989; Rothblatt et al., 1989; Sadler et al., 1989; Feldheim et al., 1992). SEC61, SEC62, and SEC63 have also been shown to interact genetically (Rothblatt et al., 1989), and the three proteins form a complex together with two other as yet unidentified proteins of 31.5 and 23 kD (Deshaies et al., 1991). These Sec proteins are intimately associated with translocating proteins. A translocation-trapped intermediate form of the yeast mating factor precursor, prepro-ot-factor (Pl~F), ~ may be chemically cross-linked to Sec61p, and to a lesser extent, Sec62p (Miisch et al., 1992; Sanders et al., 1992). Cross-linking to Sec62p was ATP inhibited, while association with Sec61p was ATP dependent (Mtisch et al., 1992). Mutations in SEC62 or SEC63 significantly reduced the degree of cross-linldng to Sec61p (Sanders et al., 1992). 1. Abbreviations used in this paper: hsc, heat-shock protein; p~F, procxfactor; ppaF, prepro-c~-factor; SRP, signal recognition particle. Translocating ppcxF also associates with the heat-shock cognate protein (hsc70) homologue, BiP, in the ER lumen (Sanders et al., 1992). Earlier studies demonstrated a re. quirement for BiP with the discovery that mutations in its gene (KAR2) caused the accumulation of a variety of untranslocated secretory preproteins (Vogel et al., 1990). Intriguingly, Sec63p has a polypeptide domain facing the ER lumen that is 42 % identical to the dnaJ protein from F~che. richia coli (Sadler et al., 1989; Feldheim et al., 1992). Because Kar2p is 50% identical to the dnaK protein from E. coli (Rose et al., 1989), and the dnaK and dnaJ proteins interact during bacteriophage replication (Yamamoto et al., 1987), it is tempting to speculate that Sec63p and Kar2p alsc interact. Exactly how Kar2p facilitates translocation remains unknown. A principal function of hsds may be to preven! protein aggregation, or to disassociate protein-protein interactions (Pelham, 1986). For example, mammalian BiP preferentially binds hydrophobic peptides (Flynn et al., 1991), and peptide binding stimulates ATP hydrolysis, which is followed by peptide release (Flynn et al., 1989). Kar2p may be an acceptor for nascent translocating proteins, keeping the emerging peptide in an unfolded conformation until translocation is complete. This would prevent aberrant or premature folding from occurring, especially that mediated by hydrophobic interactions (for reviews see Pelham, 1986; Rothman, 1989; Gething and Sambrook, 1992). Alterna. tively, by analogy to the requirement for dnaK and dnaJ during X replication (Zylicz et al., 1989), Kar2p and Sec63~ might interact during translocation to activate the translocation apparatus. Other hsc70 molecules are also involved in protein transloThe Rockefeller University Press, 0021-9525/93/01/95/8 $2.00 The Journal of Cell Biology, Volume 120, Number 1, January 1993 95-102 95 on M ay 4, 2017 D ow nladed fom Published January 1, 1993