Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex.

Abstract

Abstract. The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61ot, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61/~ and 3') to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes.To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the proteaseresistance of Sec61ot which is shielded by these ribosomes. In contrast, p34 and plS0 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes.These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process. MANY proteins are targeted to the mammalian ER membrane by means of the signal recognition particle (SRP) t and its membrane receptor (SRPreceptor or docking protein) (for review see Rapoport, 1992). They are subsequently transported across the membrane, presumably through a protein-conducting channel. The main component of this channel may be Sec61p (now named Sec61ot), a multi-spanning membrane protein, originally discovered in S. cerevisiae by genetic screening for translocation defects (Deshaies and Schekman, 1987;Stifling et al., 1992). Sec61, is adjacent to polypeptide chains passing through the ER membrane of yeast and mammals, it is essenAddress all correspondence to Dr. Tom Rapoport, Max-Delbriick-Center for Molecular Medicine, Robert-R6ssle-Street 10, 13125 Berlin-Buch, Federal Republic of Germany.The present address of Dr. Dirk G6rlich, Wellcome/Cancer Research Campaign Institute of Cancer and Developmental Biology, Tennis Court Road, CB2 1QR Cambridge, England.1. Abbreviations used in this paper: eq., equivalents; PK-RM, puromycin/high salt-stripped rough microsomes; RM, rough microsomes; SLR, S-labeling reagent; SRP, signal recognition particle; TRAM protein, translocating chain-associating membrane protein.tial for prote...

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

“…Chymotrypsin digestion can be used to selectively cleave Sec61␣ molecules that are not covered by ribosomes (Kalies et al, 1994); cleavage occurs in the loops between transmembrane segments 6 and 7 and 8 and 9 Song et al, 2000). Although only the free Sec61 population was degraded by chymotrypsin (our unpublished data), this led to the dissociation of ϳ60% of the prebound ribosomes ( Figure 5D), suggesting that Sec61 molecules that previously were beneath a ribosome became accessible to the protease.…”

“…Previous work has shown that the Sec61 complex can bind not only RNCs but also nontranslating ribosomes with nanomolar affinity (Borgese et al, 1974;Kalies et al, 1994;Prinz et al, 2000a). Ribosomes remain bound to ER membranes after nascent chain release (Adelman et al, 1973) and do not easily dissociate from the membrane (Borgese et al, 1973;Potter and Nicchitta, 2002).…”

“…In membrane proteins, TM sequences are partitioned sideways through the walls of the channel into the lipid phase, leaving some parts of the polypeptide chain in the cytosol and others in the ER lumen. For both classes of proteins, the translating ribosome stays bound to the channel during the entire translocation process (Mothes et al, 1997).Previous work has shown that the Sec61 complex can bind not only RNCs but also nontranslating ribosomes with nanomolar affinity (Borgese et al, 1974;Kalies et al, 1994;Prinz et al, 2000a). Ribosomes remain bound to ER membranes after nascent chain release (Adelman et al, 1973) and do not easily dissociate from the membrane (Borgese et al, 1973;Potter and Nicchitta, 2002).…”

“…Given that the concentration of free ribosomes in a secretory cell is ϳ0.3 M, 95% of the Sec61 binding sites should be occupied (Blobel and Potter, 1967;Ramsey and Steele, 1976;Barle et al, 1999). Indeed, electron microscopy pictures show that the ER is densely populated with membrane-bound ribosomes, and isolated rough microsomes have a high ribosome content (Adelman et al, 1973;Kalies et al, 1994). The occupation of ribosome binding sites raises the question of how ribosomes that carry nascent chains destined for translocation can efficiently be targeted to Sec61.…”

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“…S6 phosphorylation, would then contribute to the control of both protein synthesis and protein degradation. In addition to the docking protein, the binding of ribosomes to the endoplasmic reticulum is promoted by membrane receptor proteins with molecular masses of 34 kDa (Ichimura et al, 1992) and 180 kDa (Savitz & Meyer, 1993), and by the Sec61p-complex (Kalies et al, 1994). It is possible that these proteins not only control the rate of protein synthesis, but also simultaneously the activity of the autophagic pathway.…”