Soluble factors stimulating secretory protein translocation in bacteria and yeast can substitute for each other.

Abstract

mRNA for prepro-a--factor (ppc), a yeast secretory glycoprotein, was translated in a wheat germ cell-free system that was posttranslationally supplemented either with inverted vesicles from the plasma membrane ofEscherichia coli (INV) or with microsomes from Saccharomyces cerevisiae. A postribosomal supernatant (PRS) from E. coli was found to stimulate translocation of ppa across the INV membrane. A yeast PRS could substitute for its E. coli counterpart. Likewise, an E. coli PRS could substitute for a yeast PRS and stimulate translocation of ppa across yeast microsomal membranes.The processes of secretory protein translocation across the prokaryotic plasma membrane and the eukaryotic endoplasmic reticulum (ER) have common features. Most strikingly, important structural and functional properties of the signal sequences of both prokaryotic and eukaryotic secretory proteins appear to have been conserved (1)(2)(3)(4)(5)(6)(7). However, it is not known whether or to what extent the corresponding signal recognition and translocation machineries resemble each other. Although genetic studies (for review see refs. 8 and 9) and biochemical studies (10, 11) indicate the existence of such machineries in prokaryotes, none of the components have so far been isolated. Thus, it is not known whether prokaryotes contain equivalents of the signal recognition particle (SRP) and its cognate receptor in the ER, the SRP receptor (for review, see ref. 12).Recently an extensively subfractionated cell-free system from Escherichia coli was developed in which translocation across inverted vesicles of the E. coli plasma membrane (INV) was demonstrated to be stimulated by a soluble factor or factors (10). Likewise, an activity present in the cytosol of yeast (Saccharomyces cerevisiae) has recently been shown to stimulate secretory protein translocation across yeast microsomal membranes (13). In this paper, we show that these prokaryotic and eukaryotic factors can substitute for each other in a cell-free translocation system containing INV or yeast microsomal membranes.METHODS AND MATERIALS SP6 Transcription. Plasmid pDJ100 containing prepro-afactor (ppa) structural gene (14) cloned in the BamHI site of the polylinker of pSP65 (15) was a gift of David Julius (Columbia University). Plasmid pLB8000 containing the structural gene for X phage receptor (16), LamB, cloned in the Sma I site of the polylinker of pSP64 was a gift of Spencer Benson (Princeton University). Plasma pDJ100 was linearized with Xba I, and plasmid pLB8000 was linearized with EcoRI prior to transcription. Transcriptions using the SP6 phage system were done as described in ref. 15.Wheat Germ Translation. Translations in the wheat germ system were done essentially as described by Erickson and Blobel (17). ppa mRNA (200 ng per 25 ,ul of translation mix) was translated in a staphylococcal nuclease-treated wheat germ system (8 ,ul of wheat germ extract per 25 ,ul of translation mix). The concentrations of KOAc and Mg(OAc)2 were adjusted to 112 mM and 2.1 mM, respectively ...

Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle.

“…A larger (12S) putative targeting factor was characterized several years ago from E. coli and also did not appear to contain RNA (22). Recent evidence (6) suggests that the bacterial and yeast factors which stimulate secretory protein transport can substitute for each other and thus may be functionally related. Their roles in this process remain to be elucidated, but their differences in structure and localization (see above) suggest that they do not act via a mechanism similar to SRP.…”

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

“…(or to contain either an inactive form or a negligible amount of) all those cytosolic factors that have so far been shown to be involved in protein translocation across microsomal membranes (Walter and Blobel, 1980;Fecycz and Blobel, 1987;Chirico et al, 1988). In fact, we show here that nuclear-encoded mitochondrial proteins, when synthesized in a wheat germ system, are not imported into yeast mitochondria to a significant extent unless a yeast postribosomal supernatant (PRS) ~ is added to the import reaction.…”

Requirement of different mitochondrial targeting sequences of the yeast mitochondrial transcription factor Mtf1p when synthesized in alternative translation systems

“…RRL and WGE translation kits; Promega) since yeast is not a typical animal or a plant. Furthermore, it has been reported that the cell lysate from different systems exhibits importstimulating activity [25][26][27][28][29]. The potential influence of such stimulating factors may partly account for the unusual import of Mtf1p.…”