Determining the topology of an integral membrane protein.

A variety of methods have been developed for assigning the aqueous domains of integral membrane proteins to either side of a biological membrane. Once the sequence of a protein is known from its DNA sequence it is possible to study the topology of the protein. This unit provides protocols in which the water-soluble domains can be tested for their accessibility to reagents added to membranes with a defined orientation. Tagging of hydrophilic regions of the protein with different epitopes and probing of their orientation with respect to the membrane is also described. Finally, a procedure for fusion of a reporter enzyme to truncated fragments of the protein is provided. The fusion protein is used as a sensor of sequence disposition relative to the membrane.

Sec61p is the main ribosome receptor in the endoplasmic reticulum of Saccharomyces cerevisiae.

A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

Mammalian Sec61 is associated with Sec62 and Sec63.

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.

Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA.

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the protein-conducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved.

Protein translocation into the endoplasmic reticulum (ER)--two similar routes with different modes.

Protein translocation into the endoplasmic reticulum (ER) occurs cotranslationally with the ribosome tightly bound at the membrane, or post-translationally. Transport of polypeptides is performed by an elaborate structure in the ER membrane consisting of numerous proteins. Both pathways have been reconstituted in vitro using proteoliposomes that contain purified components. Primary structures of these key components have been identified and recent electron microscopic data provide a first impression of the tertiary structure of the translocation apparatus. However, the precise function of most subunits of the translocation apparatus is still a mystery and little is known about structural changes in the translocation site during a transport cycle.

The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation.

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the beta subunit (Sec61beta) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61beta is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61beta kinetically facilitates cotranslational translocation, but is not essential for it. Using chemical cross-linking we show that Sec61beta not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61beta and SPC25 and between Sec61beta and Sec61alpha depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

Binding of signal recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction.

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC-SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation.

The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Membrane topology of the 12- and the 25-kDa subunits of the mammalian signal peptidase complex.

The cleavage of signal sequences of secretory and membrane proteins by the signal peptidase complex occurs in the lumen of the endoplasmic reticulum. Mammalian signal peptidase consists of five subunits. Four have been cloned, SPC18, SPC21, SPC22/23, and SPC25, of which all but SPC25 have been demonstrated to be single-spanning membrane proteins exposed to the lumen of the endoplasmic reticulum. We have determined the cDNA sequence of the remaining 12-kDa subunit (SPC12) as well as the membrane topologies of SPC12 and SPC25 in rough microsomes. Both polypeptides span the membrane twice with their N and C termini facing the cytosol and contain only very small, if any, lumenal domains. Therefore, SPC12 and SPC25 are likely to be involved in processes other than the enzymatic cleavage of the signal sequence.

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

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 Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) 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 protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 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.

A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation.

SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.