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Signal recognition particle (SRP), an 11S ribonucleoprotein (Walter and Blobel (1982) Nature 299, 691–698), is required for translocation of secretory proteins across microsomal membranes (Walter and Blobel (1980) Proc. Natl. Acad. Sci. USA 77, 7112–7116)and for asymmetric integration into microsomal membranes of a transmembrane protein (Anderson et al., (1982) J. Cell Biol. 93, 501–506). We demonstrate here that SRP is also required for translocation of the lysosomal protease cathepsin D across microsomal membranes.

By deletion of selected segments from a bovine opsin complementary DNA clone and subsequent analysis of transcripts in a cell-free translation–translocation system, we have localized two out of four theoretically conceivable signal sequences required for the integration of opsin into microsomal membranes.

Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin. Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes. The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity.

We have cloned and sequenced cDNA for the receptor that mediates the endocytosis and transcellular transport of IgA and IgM across many glandular epithelia into external secretions. This receptor contains five extracellular domains which are strikingly homologous to each other and to immunoglobulin variable regions.

mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized α-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat α-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of α-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communislectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature α-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by α-methyl mannoside, but not to R. communislectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

Concurrently with or shortly after their synthesis on ribosomes, numerous specific proteins are unidirectionally translocated acrossor asymmetrically integrated intodistinct cellular membranes. Thereafter, subpopulations of these proteins need to be sorted from each other and routed for export or targeted to other intracellular membranes or compartments. It is hypothesized here that the information for these processes, termed “protein topogenesis,” is encoded in discrete “topogenic” sequences that constitute a permanent or transient part of the polypeptide chain. The repertoire of distinct topogenic sequences is predicted to be relatively small because many different proteins would be topologically equivalent—i.e., targeted to the same intracellular address. The information content of topogenic sequences would be decoded and processed by distinct effectors. Four types of topogenic sequences could be distinguished: signal sequences, stop-transfer sequences, sorting sequences, and insertion sequences. Signalsequences initiate translocation of proteins across specific membranes. They would be decoded and processed by protein translocators that, by virtue of their signal sequence-specific domain and their unique location in distinct cellular membranes, effect unidirectional translocation of proteins across specific cellular membranes. Stop-transfersequences interrupt the translocation process that was previously initiated by a signal sequence and, by excluding a distinct segment of the polypeptide chain from translocation, yield asymmetric integration of proteins into translocation-competent membranes. Sortingsequences would act as determinants for posttranslocational traffic of subpopulations of proteins, originating in translocation-competent donor membranes (and compartments) and going to translocation-incompetent receiver membranes (and compartments). Finally, insertionsequences initiate unilateral integration of proteins into the lipid bilayer without the mediation of a distinct protein effector. Examples are given for topogenic sequences, either alone or in combination, to provide the information for the location of proteins in any of the intracellular compartments or for the asymmetric orientation of proteins and their location in any of the cellular membranes. Proposals are made concerning the evolution of topogenic sequences and the relationship of protein topogenesis to the precellular evolution of membranes and compartments.

An anti-idiotypic antibody approach was used to identify an integral membrane protein of the chloroplast envelope as a receptor for protein import into the chloroplast stroma. The import receptor is found in contact sites between the outer and inner membrane of the chloroplast envelope.

Membrane assembly was observed to proceed in cell-free extracts. Specifically, the membrane glycoprotein of vesicular stomatitis virus was synthesized in crude extracts of wheat germ in the presence of membrane vesicles derived from pancreatic endoplasmic reticulum. The resulting glycoprotein spans the lipid bilayer asymmetrically, is glycosylated, and is indistinguishable in these respects from the form of the glycoprotein found in the rough endoplasmic reticulum of virus-infected cells. Both glycosylation and asymmetric transmembrane insertion of the glycoprotein into membranes in vitrorequire protein synthesis in the presence of membranes. The carboxyl-terminal 5% of the polypeptide chain is located on the external surface of vesicles, corresponding to the cytoplasmic surface of the endoplasmic reticulum in cells. Most, or all, of the amino-terminal portion of the glycoprotein, as well as the protein-bound carbohydrate, appears to be located within the lumen of the membrane vesicles. These findings demonstrate that insertion of this membrane protein occurs during or immediately after protein synthesis. The results are consistent with the concepts that the growing membrane protein is extruded across the endoplasmic reticulum membrane amino terminus first and that glycosylation is restricted to the lumenal surface of the membrane. The cell-free system reported here should prove valuable for studying these processes.

Transcription is regulated by the complex interplay of repressors and activators. Much of this regulation is carried out by, in addition to gene-specific factors, complexes of more general transcriptional modulators. Here we present the characterization of a novel family of transcriptional regulators in yeast. Wtm1p (WD repeat-containing transcriptional modulator) was identified as a protein present in a large nuclear complex. This protein has two homologs, Wtm2p and Wtm3p, which probably arose by gene duplications. Deletion of these genes affects transcriptional repression at several loci, including derepression of IME2, a meiotic gene normally repressed in haploid cells. Targeting of these proteins to DNA resulted in a dramatic repression of activated transcription. In common with a mutation in the histone deacetylase RPD3, wtm mutants showed increased repression at the silent mating-type locus, HMR, and at telomeres. Although all three Wtm proteins could act as transcriptional repressors, Wtm3p, which is the least homologous, appeared to have functions separate from those of the other two. Wtm3p did not appear to be complexed with the other two proteins, was essential for IME2repression, and could not efficiently repress transcription in the absence of the other Wtm proteins. These data suggested that Wtm1p and Wtm2p are repressors and that Wtm3p has different effects on transcription at different loci.

The dramatic expansion of the peroxisomal compartment known to occur in the methanol-utilizing yeast Hansenula polymorphaon transfer from glucose- to methanol-containing media was shown to be accompanied by the synthesis of at least six major polypeptides that dominate the polypeptide pattern of total cell extracts analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Two of these polypeptides have been identified by immunochemical methods as the monomers of the peroxisomal enzymes alcohol oxidase and catalase. We have studied the biosynthesis of these two peroxisomal enzymes, both by in vitrotranslation and by in vivolabeling experiments. By the criterion of mobility in NaDodSO4/polyacrylamide gel electrophoresis, the in vitro- and in vivo-synthesized monomers were indistinguishable from each other, both in the case of alcohol oxidase and in that of catalase. Thus, neither of these peroxisomal enzymes appear to be synthesized as larger precursors. However, further analysis of in vitro-synthesized versus mature peroxisomal alcohol oxidase showed that the in vitro-synthesized form sedimented as a 5S monomer and not, like the mature peroxisomal enzyme, as a 20S octamer. Moreover, the in vitro-synthesized form was highly susceptible to trypsin digestion whereas the mature 20S octamer appeared to be resistant.

A highly active in vitrosystem for the translation of globin mRNA, resulting in more than 10 rounds of translation, is described. The reconstituted system consists of native small ribosomal subunits of rabbit reticulocytes (as a source of initiation factors as well as small ribosomal subunits), large subunits derived from rat liver polysomes by the puromycin-KCl procedure, and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant. In this system no differences were found between globin messenger ribonucleoprotein and globin mRNA.

Two distinct proteins were found to be tightly bound to the heterogeneous messenger RNAs associated with polysomes in mouse L cells and rat hepatocytes. The molecular weight (78,000) of the larger of these two proteins is identical to that of the protein previously found associated with rabbit globin messenger RNA. This protein is shown to be bound to the adenylate-rich region of messenger RNA. The molecular weight of the smaller protein associated with messenger RNAs from hepatic and L-cell polysomes is similar to that found in globin messenger ribonucleoprotein.

Hepatic ribosomes have been dissociated into biologically active subunits as follows. Polysomes were treated at 0°C with puromycin at high ionic strength. This released most of the nascent polypeptide chains without dissociating the polysomes, which retained the mRNA and the tRNA moiety of peptidyl tRNA, but were unable to continue the translation of mRNA. The polysomes were then heated to 37°C, when they dissociated completely into subunits. Similar treatment without puromycin resulted in only partial dissociation.

5S RNA is removed from the large ribosomal subunit of both rat liver and rabbit reticulocyte ribosomes by treatment with EDTA to remove magnesium ions. The RNA is removed not as a naked molecule, but in association with a single ribosomal protein (molecular weight about 35,000) as a ribonucleoprotein, sedimenting at about 7 S. Conditions for the exclusive removal of the 5S RNA-protein complex have been established.Both ribosomal subunits undergo distinct changes in their sedimentation rate, concomitant with the loss of their biological activity, when sedimented into sucrose gradients containing high concentrations of monovalent ions and low concentrations of magnesium. Under these conditions the large subunit loses 5S RNA, the 5S RNA-associated protein, and several more proteins.

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl2, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [3H]puromycin into hot acid-insoluble material and from the release of [3H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (∼15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.

0.3–0.5 M MgCl2 was used to disassemble nuclei and to isolate by a single centrifugation in less than 3 hr a nuclear envelope fraction in 55–60% yield as assessed by phospholipid recovery. Its gross chemical composition was determined and its morphology was studied electron microscopically by sectioning, freeze etching, and negative staining procedures.

The puromycin-induced dissociation, at high ionic strength, of active ribosomes from reticulocytes resulted in the release of protein-free, apparently undegraded, messenger RNA for globin, which was identified by centrifugation on a sucrose density gradient. This procedure should make messenger RNA from various cells available for isolation on a large scale, and has several advantages over procedures that use detergents or magnesium chelators.