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Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis.

In Saccharomyces cerevisiae, Spo12p is involved in mitosis and is essential for meiosis. We found that Spo12p is imported into the nucleus by the karyopherin Kap121p. A complex containing Spo12p and Kap121p was isolated from cytosol and was also reconstituted with recombinant proteins, indicating that this interaction is direct. Spo12p was mislocalized to the cytosol inpse1–1, a temperature-sensitive strain harboring a mutation of Kap121p, at the permissive temperature, confirming an essential role for Kap121p in Spo12p import. Spo12p was also mislocalized in a pse1–1/pse1–1homozygous strain, suggesting it is imported via the same pathway in diploid cells. Furthermore, we found that pse1–1/pse1–1shows a sporulation defect similar to that ofspo12Δ/spo12Δ.In addition, we have characterized the Spo12p nuclear localization signal, mapped it to residues 76–130, and identified residues within this region that are important for nuclear localization signal function.

Karyopherin β2 (Kapβ2, transportin) binds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapβ2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapβ2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapβ2, indicating that Nup98 can dissociate Kapβ2 from its substrate. Binding of Kapβ2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapβ2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF bound to Nup98 at a region adjacent to the Kapβ2-binding site. These findings suggest a model where 1) import substrate is released from Kapβ2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapβ2 from Nup98 allowing repeated cycles of import.

Binding of GTP-bound Ran (RanGTP) to karyopherin β1 (Kapβ1) releases import cargo into the nucleus. Using an ultrastructural, biochemical, and functional approach, we have studied the mechanism by which Kapβ1·RanGTP is recycled at the nuclear pore complex for repeated rounds of import. In vitro, Kapβ1 bound to the RanBP1-homologous (RBH) domains of Nup358 in the presence of either RanGTP or RanGDP, forming trimeric complexes. The Kapβ1·RanGTP·RBH complex resisted dissociation by RanBP1 and GTP hydrolysis by Ran GTPase activating protein 1. Ran-dependent binding of gold-conjugated Kapβ1 to the cytoplasmic fibers of the nuclear pore complex in digitonin-permeabilized cells and RanBP1 competition confirmed the in vitrobinding data. Interaction of karyopherin α and a classical nuclear localization sequence peptide with the Kapβ1·RanGTP·RBH complex stimulated GTP hydrolysis by Ran GTPase activating protein 1 both in vitroand in permeabilized cells. This GTP hydrolysis was required for reinitiation of import of a nuclear localization sequence-bearing substrate in permeabilized cells. These data suggest that GTP hydrolysis on the RBH domains of Nup358 couples the termination of one cycle of nuclear import with the initiation of the next.

Abstract.: Plastoglobules are conspicuous lipid-containing structures in the chloroplast stroma and are thought to serve as lipid reservoirs for thylakoid membranes. Plastoglobules also contain low levels of proteins. We have purified plastoglobules from a pea chloroplast membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified plastoglobules revealed more than a dozen distinct polypeptides that we propose to term plastoglobulins. For one of the proteins, termed plastoglobulin 1 (PG1), we have obtained partial N-terminal and internal protein sequences. The amino acid sequence, deduced from cDNA, encoded a precursor protein of a calculated mass of 38,491 Da which contained a 48-residue-long N-terminal signal sequence. Pre-PG1 obtained by coupled in-vitro transcription/translation was imported into pea chloroplasts, its signal sequence was cleaved and mature PG1 was assembled into plastoglobules. Homology searches of the data bases revealed similarity of PG1 to the plastoglobule-associated protein of Capsicum annuum, the carotenoid-associated protein of Cucumis sativus and to a protein of the cyanobacterium Synechocystis sp., indicating that PG1 is a novel member of an ancient protein family. Immunoelectron microscopy using PG1-specific antibodies indicated that PG1 is present in multiple copies on the surface of plastoglobules.

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.

A yeast cytosol is shown to contain two distinct activities that stimulate protein translocation across microsomal membranes. One activity was purified. It consists of two constitutively expressed 70K heat shock related proteins that increase the rate of translocation. Possible mechanisms of action of these proteins are discussed.

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.

In addition to its previously characterized, six different polypeptide components, signal recognition protein—which functions in protein translocation across and integration into the endoplasmic reticulum membrane—contains a 7S RNA molecule. The RNA is closely identified with the small cytoplasmic 7SL RNA and is required for both structural and functional properties of signal recognition protein—which we therefore rename signal recognition particle.

Ovalbumin is shown to contain an internal rather than an amino-terminal signal sequence. This internal signal can be recovered in a tryptic fragment which comprises residues 229–276 of mature ovalbumin and contains a region of striking sequence homology to amino-terminal cleaved signals of two other oviduct secretory proteins. The isolated tryptic fragment is used as a probe to demonstrate that a signal receptor is present in membrane vesicles derived from the rough but not in those derived from the smooth endoplasmic reticulum.

The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell λ expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e.Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.

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 a rat cDNA whose deduced primary structure yields a protein of 117.4 kDa. Because this protein contains RNA helicase consensus motifs, among them a “DEAD” box, we have termed it HEL117 (for helicase of 117.4 kDa). Besides the helicase consensus motifs, HEL117 contains an arginine-serine (RS)-rich domain, which occurs in some proteins involved in RNA splicing. Moreover, the COOH-terminal region of 78 residues of HEL117 is 38.5% identical and 59% similar to the COOH-terminal region of a yeast PRP5 protein that is involved in RNA splicing. Rabbit antibodies generated against a synthetic peptide of HEL117 identified a single polypeptide not only in rat cells but also in cells of other mammals as well as chicken. The antibodies revealed a finely punctate and speckled intranuclear staining in immunofluorescence microscopy. A monoclonal antibody against a human splicing factor containing an RS domain (SC35) showed, in double immunofluorescence microscopy, largely overlapping staining consistent with HEL117 being involved in RNA splicing.

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.

Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin. Yeast contain a homologue of karyopherin α named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I. To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin α homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera. Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose. Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII. Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin β. The yeast α and β karyopherin subunits were expressed in Escherichia coliand were purified. When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells. We propose that all karyopherins function as α/β heterodimers.

At least one essential function of Smt3p, aSaccharomyces cerevisiaeubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub∼Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin α, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitrobinding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+PICs was dependent on karyopherin β and was blocked by antibodies to β. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.

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.

Canine pancreas rough microsomes were solubilized in a high salt buffer containing sodium cholate, a detergent extract prepared by high speed centrifugation, and vesicles were reconstituted from the extract by a detergent dialysis procedure. The reconstituted vesicles, lacking resident lumenal proteins, translocated in vitro-synthesized preprolactin in a cotranslational, SRP-dependent manner. The translocated precursor was processed to mature prolactin and protected from digestion by exogenous protease. Vesicles were also reconstituted from detergent extracts depleted of glycoproteins by chromatography on concanavalin-Sepharose. The depleted vesicles, containing a full complement of SRP receptor but deficient in the glycoprotein subunit of signal peptidase, the ribophorins, and other glycoprotein components, were functional in the targeting and binding of nascent preprolactin but exhibited a greatly diminished capacity for translocation.

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.

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound 35S-labeled mammalian karyopherin β, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin β), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.

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.

The heterodimeric karyopherin functions in targeting a nuclear localization sequence (NLS)-containing protein to the nuclear pore complex followed by Ran-GTP and p10-mediated translocation of the NLS protein into the nucleoplasm. It was shown recently that Ran-GTP dissociated the karyopherin heterodimer and, in doing so, associated with karyopherin β (Rexach, M., and Blobel, G.(1995) Cell83, 683-692). We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin β binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the Ran-specific GTPase activating protein). Inhibition of RanGAP-stimulated GTP hydrolysis by karyopherin β was dependent on karyopherin β concentration relative to Ran-GTP. Complete inhibition of RanGAP was observed at karyopherin β concentrations that were equimolar to Ran-GTP. In gel filtration experiments, we found Ran-GTP and karyopherin β to form a stoichiometric complex. Ran-GDP bound only weakly to karyopherin β. We propose that stoichiometric complex formation between karyopherin β and Ran-GTP renders Ran-GTP inaccessible to RanGAP.

We previously showed that RanGTP forms a 1:1 complex with karyopherin β that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G. (1996) J. Biol. Chem.271, 5313–5316) and karyopherin β functionally inactive (Rexach, M., and Blobel, G. (1995) Cell83, 683–692). Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin β complex. Here we show using BIAcore™, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin β and RanGTP are recycled efficiently in a reaction that involves karyopherin α, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyopherin α first releases RanGTP from karyopherin β in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin αβ binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin αβ stimulate recycling of karyopherin β and Ran in a manner similar to the C terminus of Nup1.

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.

The capacity of the rough endoplasmic reticulum (RER) membrane of eukaryotic cells to translocate nascent presecretory proteins from the cytosol to the intracisternal space is preserved on cell fractionation and can be assayed in vitro1. Two attempts to characterize this translocation activity have been reported. Warren and Dobberstein2reported that microsomal membranes can be depleted of their translocation activity by extraction with a solution of high ionic strength (500 mM KCl) and that activity can be restored to the depleted membranes by re-addition of the salt extract. On the other hand, Walter et al.3reported that KCl extraction of the microsomal membrane does not result in complete depletion of its translocation activity. However, mild trypsinization of the microsomal membrane released a tryptic fragment(s) from the membrane which, when recombined with a tryptically inactivated membrane fraction, restored translocation activity3. We now show that both the trypsin and the KCl extracted factors, but not the membrane-integrated remainder of the translocation apparatus, contain at least one sulphydryl group that is essential for activity.

The LamB protein is an integral membrane protein of the outer membrane of Escherichia coli. We have now found that, when synthesized in an E. coli cell-free translation system supplemented with inverted vesicles derived from the E. coli inner membrane, LamB protein is integrated into the vesicle membrane as assayed by its resistance to extraction at alkaline pH. These data suggest that the inner membrane is the primary site for integration of LamB protein prior to subsequent sorting to the outer membrane. When synthesized in a wheat germ cell-free translation system supplemented with canine microsomal membranes, LamB protein is glycosylated at one or two cryptic sites, and surprisingly, it is translocated across instead of being integrated into the vesicle membrane. We suggest that the translocation machinery of the microsomal membrane, although able to recognize the signal sequences) of LamB, is unable to recognize its stop-transfer sequences), thereby yielding translocation instead of integration.

The calcium pump protein (CPP) or Ca2+-ATPase is the predominant integral membrane protein of the sarcoplasmic reticulum (SR) of skeletal muscle1,2. CPP is a single polypeptide chain of molecular weight (MW) 119,000 (ref. 3) which loops in and out of the membrane at least three times4–8. Recently, a number of integral transmembrane proteins possessing a single hydrophilic domain on each side of the membrane have been observed to use a signal sequence, cleaved9,10or uncleaved11, to initiate translocation of their ectoplasmic domain12. Microsomal membranes when present during translation are capable of correctly integrating these de novo synthesized proteins into the membrane9,11. We have used this in vitro translocation system to investigate the integration of CPP into the microsomal membrane. We found that CPP is synthesized without a cleaved signal sequence and that it can be integrated into heterologous microsomal membranes only when these are present during translation, but not when they are present after completion of translation.