Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.

Abstract

The capacity of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer can be largely abolished by extracting them with high ionic strength buffers. It can be reconstituted by adding the salt extract back to the depleted membranes [Warren, G. & Dobberstein, B. (1978) Nature plex. The synthesis of secretory and several membrane proteins so far investigated is thought to be initiated on free ribosomes (1, 2). When the nascent polypeptide chain emerges from the ribosome, a specific section of it, the signal sequence, was proposed to interact with receptors in the endoplasmic reticulum (ER), triggering the formation of a ribosome-membrane junction and initiating the translocation process of the nascent chain into the cisternae of the ER (1, 2). In most cases the signal sequence is located at the NH2-terminal portion of the nascent chain (2) and is cleaved by signal peptidase (3,4), presumably on the luminal side of the ER (5, 6), concomitantly with elongation and translocation of the protein.Little is known about the recognition and transport machinery that is involved in the translocation process. The translocation activity of the microsomal vesicles has been assayed directly, after extraction and reconstitution experiments. One approach used salt extraction (7); the other one used limited tryptic dissection (6) of the microsomes. In both cases, a water soluble fraction and a translocation-inactive membrane fraction were obtained that recombined to reconstitute the translocation activity. Both water soluble fractions have been shown to be functionally equivalent (8) (the trypsin-derived fraction reconstitutes salt-depleted membranes and vice versa) and both contain a sulfhydryl group, which is required for their activity (8).We describe here the purification to homogeneity of the active component of the salt extract that reconfers translocation activity to the salt-extracted microsomal membrane vesicles. METHODS All preparative procedures were carried out at 0-40C. Optical absorbance determinations were done in 1% NaDodSO4. All glassware was siliconized. The 4 M KOAc stock solution was adjusted to pH 7.5. Preparation of Microsomal Membranes. Rough microsomal membranes were prepared from freshly excised dog pancreas (9) as described (10), with the following modifications: all buffers contained 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride. The rough microsomes were collected by centrifugation for 2.5 hr at 140,000 X gavg The resulting pellet of rough microsomes (RM) was resuspended by manual homogenization in a Dounce homogenizer (A-pestle) in buffer A (250 mM sucrose/50 mM triethanolamine-HOAc, pH 7.5 [(EtOH)3N-HOAc]/1.0 mM dithiothreitol) to a concentration of 50 A280 units/ml.Adsorbed ribosomes and proteins were removed by passing the membranes through a Sepharose C1-2B column (11) in a low-salt buffer [50 mM (EtOH)3N-HOAc/0.5 mM Mg(OAc)2/1 mM dithiothreitol]. RM (20 ml) were loaded on a 200-ml column (upward flow, 15 ml/hr). The turbid fractions wer...

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

“…The dissociation constant of SRP9/14 and SRP RNA is smaller than 0+1 nM (Janiak et al+, 1992), corresponding to a total free binding energy of more than 13+7 kcal/mol, and our preliminary estimates indicate that the closed conformation is only about 2+3-2+7 kcal/mol more stable than the open conformation+ This raises the questions of how readily reversible is the conformational switch between the open ((46 Å ϫ 25 Å) ϫ 95 Å in length) and closed ((46 Å ϫ 50 Å) ϫ 59 Å in length) Alu RNA and whether the switch could be triggered in the physiological context of SRP interacting with other cellular components such as the ribosome+ There is experimental support for the reversibility of 39 domain binding in the flexibly linked SA86 RNP, because purified compact monomers are found to crystallize as SA88 RNP-like domain-swap dimers of the open form (O+ Weichenrieder and S+ Cusack, unpubl+ data), presumably favored by the high concentration+ Also in the context of purified, complete SRP, there are experimental conditions under which the SRP9 cysteines are preferentially accessible to alkylation, indicating an opening of the Alu domain (Walter & Blobel, 1980)+…”

“…The signal recognition particle (SRP; Walter & Blobel, 1980), like the ribosome, is a cytoplasmic ribonucleoprotein particle (RNP) of ancient evolutionary origin (Poritz et al+, 1990;Bhuiyan et al+, 2000)+ SRP has an intrinsic affinity for ribosomes (Walter et al+, 1981) and its catalytic promotion of the cotranslational mode of protein translocation across membranes is well documented (Walter & Johnson, 1994;Lütcke, 1995)+ In mammals, SRP consists of the highly base-paired 300-nt-long SRP RNA and six proteins: SRP54, SRP19, and the heterodimers SRP68/72 and SRP9/14 (Fig+ 1)+ SRP9/14 associates with the terminal sequences of SRP RNA, forming the enzymatically separable Alu domain of SRP (Gundelfinger et al+, 1983), whereas the other proteins together with the central RNA sequence form the S-domain of SRP+ High resolution crystal structures are now available for a number of SRP components: the NG-and M-domains of SRP54 (Freymann et al+, 1997;Montoya et al+, 1997;Keenan et al+, 1998;Clemons et al+, 1999) and the M-domain in complex with helix 8 of SRP RNA (Batey et al+, 2000) as well as the free helices 6 (Wild et al+, 1999) and 8 (Jovine et al+, 2000) of SRP RNA, free SRP9/14 (Birse et al+, 1997), and, most recently, the Alu domain with SRP9/14 clamping together in its concave beta-sheet the 59 and 39 domains of Alu RNA (Weichenrieder et al+, 2000)+ In electron micrographs the particle appears as a flexible, tri-segmented rod of 60 Å by 260-280 Å with the two domains distinguishable at opposite ends (Andrews et al+, 1985(Andrews et al+, , 1987+ SRP selects ribosomes displaying the N-terminal signal sequence of nascent secretory and membrane proteins that first emerges at the exit pore on the large ribosomal subunit+ The Alu domain of SRP is responsible for retarding the elongation of these proteins once their export signal sequence is bound by the S-domain of SRP and prior to engagement with the translocation machinery in the endoplasmic reticulum+ Considering the apparent length of the particle, it has been pro-posed that the Alu domain might reach the cleft between the two ribosomal subunits where the elongation factors bind (Andrews et al+, 1987;Siegel & Walter, 1988), but the Alu RNP crystal structure does not support the hypothesis that elongation arrest is caused by a mechanism of mimicry-based active competition with elongation factors (Weichenrieder et al+, 2000)+ Knowledge of the preferred orientation and the degree of flexibility of the Alu domain (and the crucial C-termi...…”

Antibody to signal recognition particle in polymyositis

“…The signal recognition particle (SRP) is a complex of a unique RNA (labeled 7SL) (22,23) and 6 polypeptides with molecular weights of 72 kd, 68 kd, 54 kd, 19 kd, 14 kd, and 9 kd (24)(25)(26)(27). This cytoplasmic particle recognizes the signal sequence of secretory, lysosomal, and membrane proteins, binding to it via the 54-kd protein (28)(29)(30)(31)(32), and targets the nascent chains of these proteins to the endoplasmic reticulum by binding to a specific receptor (28)(29)(30)(31)(32)(33).…”

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein.

“…Certain peptides can act as signals to localize proteins to particular organelles such as the ER, the mitochondria (Verner and Schatz, 1988) and the nucleus (Silver and Hall, 1988). Several proteins have been identified that mediate the recognition of ER-destined proteins and their subsequent translocation across or assembly into the ER membrane (Walter and Blobel, 1980;Meyer et al, 1982;Tajima et al, 1986; Wiedrnan et al, 1987). Receptors have been proposed for mitochondrial signal peptides (Pfaller and Neupert, 1987;Pfanner et al, 1987) and recently a receptor for protein import into chloroplasts has been identified (Pain et al, 1988).…”