Hepatic membrane proteins involved in ribosome binding: identification by three procedures.

Rat liver ribosomes, isolated from rough-surfaced endoplasmic reticulum using non-ionic detergent in the presence of 25 mM KCl, were associated with non-ribosomal proteins, presumably of membranous origin. These proteins could be isolated by extracting such ribosome fractions with either deoxycholate or non-ionic detergents at higher concentrations of KCl. Analysis of the extracts by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed the presence of a number of discrete polypeptides having the following approximate molecular weights: 166,000, 107,000, 100,000, 65,000 and 36,000. Ribosomes associated with the membrane-derived proteins reattached to degranulated membranes in vitro less well than did ribosomes prepared in ways which removed the proteins. Extraction of a set of similar proteins from degranulated endoplasmic reticulum by treatment with buffered 1 M urea, also interfered with ribosome reattachment. A third approach to the identification of proteins associated with ribosome attachment sites involved the labelling with radioactive succinic anhydride of apparently similar proteins in degranulated membranes, after prior treatment of the latter, before removal of bound ribosomes, with unlabelled reagent. The results indicate that certain membrane proteins may be part of the receptor sites for binding of ribosomes to the endoplasmic reticulum in rat liver.     

Proteins, associated with 50S-ribosomal subunits of Escherichia coli MRE600]

The proteins associated with the ribosomal subunits having the molecular masses from 158 to 47 kDa were isolated from hyaloplasmic, nucleoid and membrane fractions of Escherichia coli MRE600 cells. The proteins are eliminated from 50S subunits of ribosomes by thrice washing with the 1 M ammonium chloride buffer. 50S subunit proteins were found to be immunologically related to the inner membrane proteins. The native 50S subunits of ribosomes possess the expressed ATP-ase activity, while the washed off subunits lose it completely.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli.

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

The in vitro reconstitution of rough endoplasmic reticulum membrane derived from human placenta

The isolation of rough endoplasmic reticulum from human placenta is described. Puromycin facilitated the detachment of the majority of the ribosomes from the membrane. Ribosomes could be re-attached to the stripped membrane, and puromycin was also necessary for detaching the rebound ribosomes, indicating that peptidyl-tRNA anchors the ribosomes to the membrane in both the native and the reconstituted rough membrane. Peptides labeled in-vitro on membrane bound ribosomes (native and reconstituted rough membrane) remained associated with the membrane after the ribosomes were detached. A protein with an electrophoretic mobility in SDS gels identical to that of HPL is among the membrane associated nascent proteins of the native and the reconstituted rough membrane.     

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Cotranslational secretion of diphtheria toxin and alkaline phosphatase in vitro: involvement of membrane protein(s).

Crude messenger ribonucleic acid fractions isolated from Corynebacterium diphtheriae and Escherichia coli were translated in an E. coli in vitro protein-synthesizing system and yielded precursors of the secreted proteins diphtheria toxin and alkaline phosphatase, respectively. Addition of inverted E. coli inner membrane vesicles to the system during the initial stages of translation resulted in the intravesicular segregation of mature diphtheria toxin and alkaline phosphatase. Outer membrane vesicles or inner membrane vesicles whose cytoplasmic surfaces had been treated with pronase could not mediate transmembrane transfer of diphtheria toxin or alkaline phosphatase. However, inner membrane vesicles isolated from E. coli spheroplasts which had been treated with pronase and inner membrane vesicles complexed with ribosomes during pronase treatment were functional in transmembrane transfer. At temperatures below the phase transition of E. coli membranes, no intravesicular segregation of alkaline phosphatase or diphtheria toxin was observed. The precursor forms of each protein accumulated free from the vesicles. These results suggest that an inner membrane protein, exposed on the cytoplasmic surface, plays an integral role in secretion.     

Mdm38 is a 14-3-3-like receptor and associates with the protein synthesis machinery at the inner mitochondrial membrane

Mitochondrial ribosomes synthesize core subunits of the inner membrane respiratory chain complexes. In mitochondria, translation is regulated by mRNA‐specific activator proteins and occurs on membrane‐associated ribosomes. Mdm38/Letm1 is a conserved membrane receptor for mitochondrial ribosomes and specifically involved in respiratory chain biogenesis. In addition, Mdm38 and its higher eukaryotic homolog Letm1, function as K⁺/H⁺ or Ca²⁺/H⁺ antiporters in the inner membrane. Here, we identify the conserved ribosome‐binding domain (RBD) of Mdm38 and determine the crystal structure at 2.1 Å resolution. Surprisingly, Mdm38^RBD displays a 14‐3‐3‐like fold despite any similarity to 14‐3‐3‐proteins at the primary sequence level and thus represents the first 14‐3‐3‐like protein in mitochondria. The 14‐3‐3‐like domain is critical for respiratory chain assembly through regulation of Cox1 and Cytb translation. We show that this function can be spatially separated from the ion transport activity of the membrane integrated portion of Mdm38. On the basis of the phenotypes observed for mdm38Δ as compared to Mdm38 lacking the RBD, we suggest a model that combining ion transport and translational regulation into one molecule allows for direct coupling of ion flux across the inner membrane, and serves as a signal for the translation of mitochondrial membrane proteins via its direct association with the protein synthesis machinery.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precursor pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [³⁵S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s.The [³⁵S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30–120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation.Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes.

We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes.     

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.     

Biogenesis of endoplasmic reticulum membrane in rat liver cells. III. Biosynthesis of NADPH-cytochrome c reductase and cytochrome b5 by loosely-bound ribosomes.

Membrane-bound ribosomes were separated into two distinct classes (loosely-bound and tightly-bound ribosomes) by treatment with 0.6 M KCl, 1 mM puromycin, 0.05% DOC, or 10 mM EDTA. It was also confirmed that any one of these reagents except for EDTA dissociated the same class of ribosomes from the membrane. A population of lighter microsomal vesicles was formed from rough microsomes upon the dissociation of loosely-bound ribosomes by treatment with these chemicals. Rough microsomes were subfractionated into lighter and heavier fractions, L-rMs and H-rMs, by centrifugation using a discontinuous gradient of sucrose consisting of 1.3 M, 1.5 M, and 2.1 M solutions. It was found that L-rMs was rich in loosely-bound ribosomes, whereas H-rMs contained a high proportion of tightly-bound ribosomes. It is likely that loosely-bound and tightly-bound ribosomes are heterogeneously distributed among rough microsomal vesicles. Loosely-bound ribosomes and tightly-bound ribosomes synthesize different kinds of proteins. Two microsomal membrane proteins, NADPH-cytochrome c reductase and cytochrome b5, were exclusively synthesized by loosely-bound ribosomes, whereas serum albumin, which is a major component of the secretory proteins of hepatocytes, was synthesized only by tightly-bound ribosomes. Since the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 are released from bound ribosomes to the cytoplasmic surface of endoplasmic reticulum, while those of secretory proteins are discharged into the lumen across the membrane, the strength of the association between ribosomes and microsomal membrane seems to be correlated with the direction of release of nascent peptides.     

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.     

Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes

Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane- associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized differing lengths of the nascent polypeptide. This analysis has revealed that nascent chain insertion into the membrane begins at distinct points for different presecretory proteins.     

Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria

The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane. Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase. This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.     

Stepwise reassembly of the nuclear envelope at the end of mitosis

The nuclear envelope consists of three distinct membrane domains: the outer membrane with the bound ribosomes, the inner membrane with the bound lamina, and the pore membrane with the bound pore complexes. Using biochemical and morphological methods, we observed that the nuclear membranes of HeLa cells undergoing mitosis are disassembled in a domain-specific manner, i.e., integral membrane proteins representing the inner nuclear membrane (the lamin B receptor) and the nuclear pore membrane (gp210) are segregated into different populations of mitotic vesicles. At the completion of mitosis, the inner nuclear membrane- derived vesicles associate with chromatin first, beginning in anaphase, whereas the pore membranes and the lamina assemble later, during telophase and cytokinesis. Our data suggest that the ordered reassembly of the nuclear envelope is triggered by the early attachment of inner nuclear membrane-derived vesicles to the chromatin.     

Activity of Thylakoid-bound Ribosomes in Pea Chloroplasts

Pea (Pisum sativum) chloroplast thylakoid membranes were prepared by washing in hypotonic buffers. These membranes contained bound ribosomes which were active in protein synthesis when supplemented with soluble components from a strain of Escherichia coli low in ribonuclease. After dissolving the membranes by Triton and purification of the ribosomes, sucrose density gradient profiles indicated the presence of polysomal material as well as monomeric ribosomes. Most of the products of protein synthesis remained associated with the thylakoid membranes even after ribosomes were removed completely by high salt concentrations in the absence of Mg²⁺. Of the newly formed products, 50% could be digested by pronase, while the remainder were protected by their association with the thylakoid membranes. The products are likely to be a mixture of intrinsic and extrinsic membrane proteins, with only the former completely protected by the membranes from attack by proteases.     

The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.     

BiP Modulates the Affinity of Its Co-chaperone ERj1 for Ribosomes

Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.