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The heteromeric Sec61 complex is composed of (alpha), beta and (gamma) subunits and forms the core of the mammalian ER translocon. Oligomers of the Sec61 complex form a transmembrane channel where proteins are translocated across and integrated into the ER membrane. We have studied the subcellular localisation of the Sec61 complex using both wild-type COS1 cells and cells transfected with GFP-tagged Sec61(alpha). By double labelling immunofluorescence microscopy the GFP-tagged Sec61(alpha) was found in both the ER and the ER-Golgi intermediate compartment (ERGIC) but not in the trans-Golgi network. Immunofluorescence studies of endogenous Sec61beta and Sec61(gamma) showed that these proteins are also located in both the ER and the ERGIC. Using the alternative strategy of subcellular fractionation, we have shown that wild-type Sec61(alpha), beta and (gamma), and GFP-tagged Sec61(alpha), are all present in both the ER and the ERGIC/Golgi fractions of the gradient. The presence of the Sec61 subunits in a post-ER compartment suggests that these proteins can escape the ER and be recycled back, despite the fact that none of them contain any known membrane protein retrieval signals such as cytosolic di-lysine or di-arginine motifs. We also found that another translocon component, the glycoprotein TRAM, was present in post-ER compartments as demonstrated by subcellular fractionation. Our data indicate that the core components of the mammalian ER translocon are not permanently resident in the ER, but rather that they are maintained in the ER by a specific retrieval mechanism.

We have studied the DNA of individuals who express an altered sialoglycoprotein beta on their red cells by using Southern blotting with sialoglycoprotein-beta cDNA probes. Individuals of the Leach phenotype do not express any beta (sialoglycoprotein beta) or gamma (sialoglycoprotein gamma) on their red cells, and we show that about 7 kb of DNA, including the 3′ end of the beta gene, is deleted in this DNA. Any protein product of this gene is likely to lack the membrane-associating domain of beta. We have also examined the DNA of two types of other individuals (Yus-type and Gerbich-type) who have red cells that lack beta and gamma, but contain abnormal sialoglycoproteins related to beta. These two types of DNA contain different internal deletions of about 6 kb in the beta gene. We suggest that these deletions result from the presence of two different sets of internal homology in the beta gene, and on this basis we propose structures for the abnormal Yus-type and Gerbich-type sialoglycoproteins which are consistent with the other evidence that is available. We provide evidence that beta and gamma are products of the same gene and suggest a possible mechanism for the origin of gamma based on leaky initiation of translation of beta mRNA.

We have isolated cDNA clones corresponding to the human erythrocyte membrane sialoglycoprotein beta. The clones encompass the coding region for the protein, 120 residues of the 5′ non-coding region and the 3′ non-coding region. The cDNA sequence suggests that sialoglycoprotein beta is not translated with the cleaved N-terminal signal sequence usual in a membrane protein of this type. Sialoglycoprotein beta or a closely related homologue is present in human kidney as well as erythroid cells.

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.

The lumen of the endoplasmic reticulum contains a number of distinct molecular chaperones and folding factors, which modulate the folding and assembly of newly synthesized proteins and protein complexes. A subset of these luminal components are specific for glycoproteins, and, like calnexin and calreticulin, the thiol-dependent reductase ERp57 has been shown to interact specifically with soluble secretory proteins bearing N-linked carbohydrate. Calnexin and calreticulin also interact with glycosylated integral membrane proteins, and in this study we have examined the interaction of ERp57 with these substrates. As with soluble proteins, the binding of ERp57 to an integral membrane protein is dependent upon the protein bearing an N-glycan that has undergone glucose trimming. Furthermore, ERp57 binds to newly synthesized glycoproteins in combination with either calnexin or calreticulin. We propose that ERp57 acts in concert with calnexin and calreticulin to modulate glycoprotein folding and enforce the glycoprotein specific quality control mechanism operating in the endoplasmic reticulum.

We have investigated the molecular details of the membrane insertion of the multiple-spanning membrane protein opsin. Using heterobifunctional cross-linking reagents the endoplasmic reticulum (ER) proteins adjacent to a series of defined translocation intermediates were determined. Once the nascent opsin chain reaches a critical minimum length Sec61alpha is the major ER component adjacent to the polypeptide. Using a homobifunctional reagent, the cross-linking partners from a single cysteine residue in the nascent chain were analyzed. This approach identified chain length-dependent cross-linking products between nascent opsin and a 21-kDa ribosomal protein, followed by Sec61beta and finally with Sec61alpha. Our data support a model where the sequential transmembrane domains of a multiple-spanning membrane protein are integrated at an ER insertion site similar to that mediating the insertion of single-spanning membrane proteins.

Calnexin is an integral membrane protein that acts as a chaperone during glycoprotein folding in the endoplasmic reticulum. Cross-linking studies were carried out with the aim of investigating the interactions of calnexin with glycoproteins in vitro. A truncated version of the integral membrane glycoprotein Glut 1 (GT155) was synthesized in a rabbit reticulocyte translation system in the presence of canine pancreatic microsomes. Following immunoprecipitation with an anticalnexin antiserum, a cross-linker-independent association was observed between GT155 and calnexin. In addition, the anti-calnexin antiserum immunoprecipitated a UV-dependent cross-linking product consisting of GT155 and a protein of approximately 60 kDa designated CAP-60 (calnexin-associated protein of 60 kDa). Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155, a characteristic of many calnexin interactions. A GT155 mutant that was not glycosylated (AGGT155) did not associate with calnexin or CAP-60. Calreticulin, the soluble homologue of calnexin, was also shown to interact with GT155 only when the protein bore a correctly modified oligosaccharide group. Thus, our data show that both calnexin and calreticulin with Glut 1 in a glycosylation-dependent manner.

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5′ and 3′ flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.

1. We have isolated cDNA clones corresponding to the red cell membrane anion-transport protein (Band 3). 2. The cDNA clones cover 3475 bases of the mRNA and contain the entire protein-coding region, 150 bases of the 5′ untranslated region and part of the 3′ non-coding region, but do not extend to the 3′ end of the mRNA. 3. The translated protein sequence predicts that the human red cell anion transporter contains 911 amino acids. 4. The availability of the amino acid sequence allows the interpretation of some of the many studies on the chemical and proteolytic modification of the human protein aimed at examining the structure and mechanism of this membrane transport protein.