An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.     

Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland

The occurrence of phosphorylated secretory proteins such as caseins and vitellogenin and the recent characterization of phosphorylated proteoglycans, in the xylose and protein core, has raised the question of where in the cell and how this phosphorylation occurs. Previous studies have described a casein kinase activity in the lumen of the Golgi apparatus and this organelle as the site of xylose addition to the protein core of proteoglycans. We now report the translocation in vitro of ATP into the lumen of rat liver and mammary gland Golgi vesicles which are sealed and have the same membrane topographical orientation as in vivo. The entire ATP molecule was translocated into the lumen of the Golgi vesicles; this was established by using ATP radiolabeled with tritium in the adenine and gamma-32P. Translocation was temperature dependent and saturable, with an apparent Km of 0.9 microM and Vmax of 58 pmol/mg protein/min. Preliminary evidence suggests that translocation of ATP into the vesicles' lumen is coupled to exit of AMP from the lumen. Following translocation of ATP into the lumen of the vesicles, proteins were phosphorylated.     

Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast

BiP/GRP78 is an essential member of the HSP70 family that resides in the lumen of the endoplasmic reticulum. In yeast, BiP/GRP78 is encoded by the KAR2 gene. A temperature sensitive mutation was isolated in KAR2 and found to cause a rapid block in protein secretion. Secretory precursors of a number of proteins (invertase, carboxypeptidase Y, alpha-factor, and BiP) accumulated that were characteristic of a block in translocation into the lumen of the ER. Protease protection experiments confirmed that the precursors accumulated on the cytoplasmic side of the ER membrane. Moreover, depletion of wild-type KAR2 protein also resulted in a block in translocation of secretory proteins. These results implicate BiP/GRP78 function in the continued translocation of proteins into the lumen of the ER.     

Topography of glycosylation reactions in the rough endoplasmic reticulum membrane

The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of [3H]UDP-glucose and UDP-[14C]glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.     

Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae

Isolation and biochemical analysis of the components involved in protein translocation into the rough endoplasmic reticulum (ER) requires starting material highly enriched in membranes derived from this organelle. We have chosen to study the yeast Saccharomyces cerevisiae in order to profit from the ease of genetic manipulation. To date, however, no efficient scheme has been devised that allows the purification of functional rough ER-derived membranes from yeast, largely because proteins have yet to be identified that are rough ER-specific. In the experiments described here, we expressed the human rough ER marker ribophorin I to facilitate the analysis of subcellular fractionation. We found that the endoplasmic reticulum of yeast could be separated into two distinct domains by fractionation on continuous sucrose gradients. This procedure revealed a bimodal distribution of ER markers. The yeast homologue of the heavy chain-binding protein, BiP (encoded by the KAR2 gene), and the product of the SEC62 gene were present in two fractions having equilibrium densities of 1.146 and 1.192 g/ml, respectively. In contrast, our analysis showed that preprotein translocation activity and retention of the rough ER-specific protein ribophorin I were specific only to the membrane fraction with an equilibrium density of 1.192 g/ml. To prepare fractions highly enriched in translocation competent rough ER-derived membranes for analysis, we developed a density shift fractionation scheme that optimizes the purity of membranes containing human ribophorin I. Membranes obtained by this method were found to possess the majority of the appropriate functional markers, including ATP-independent preprotein binding, ribosome binding, and post-translational translocation. Mitochondria, the major contaminant of the 1.192 g/ml fraction, were significantly depleted in density-shifted membrane populations.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

A complex of chaperones and disulfide isomerases occludes the cytosolic face of the translocation protein Sec61p and affects translocation of the prion protein

Secretion of newly synthesized proteins across the mammalian rough endoplasmic reticulum (translocation) is supported by the membrane proteins Sec61p and TRAM, but may also include accessory factors, depending on the particular translocation substrate. Studies designed to investigate the binding of anti-peptide antibodies to the carboxyl terminus of the alpha-subunit of Sec61 (Sec61palpha) lead us to the isolation of a complex of proteins that occlude the cytosolic face of Sec61palpha in microsomes that have been prepared by standard protocols used to study translocation in vitro [Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93]. This complex was shown by nanospray tandem mass spectrometry to be composed of protein disulfide isomerase (PDI), calcium binding protein 1 (CABP1/P5), 72 kDa endoplasmic reticulum protein (ERp72), and BiP (heat shock protein A5/HSPA5), and has been named TR-PDI for "translocon-resident protein disulfide isomerase complex". This constitutes a novel location for these proteins, which are known to be major constituents of the lumen of the rough endoplasmic reticulum. We have not established the function of TR-PDI at this location, but did observe that the absence of this complex results in a relative loss of correct topology of prion protein insertion across RER membranes, indicating the possibility of a functional role in vivo.     

Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean

Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.     

Yeast ribosomes bind to highly purified reconstituted Sec61p complex and to mammalian p180

To determine whether the yeast Sec61p translocation pore is a high-affinity ribosome receptor in the endoplasmic reticulum, we isolated the Sec61p complex using an improved protocol in which contaminants found previously to be associated with the complex are absent. The purified complex, which contains Sec61p with an amino terminal hexahistidine tag, was active since it rescued a sec61–3 post-translational translocation defect in a reconstituted system. Co-reconstitution of the Sec61p and Sec63p complexes into liposomes failed to support post-translational translocation, suggesting that Sec62p is required for this process. By Scatchard analysis, the purified Sec61p complex bound to yeast ribosomes when reconstituted into liposomes with a K_D of 5.6 n m , a value similar to the K_D obtained when ribosome binding to total microsomal protein was measured (2.7 n m ). In addition, a mammalian protein, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this strain exhibited ∼2.3-fold greater binding to yeast ribosomes. Despite this increase in ribosome binding, neither co- nor post-translational translocation was compromised in vivo . In sum, our data suggest that the Sec61p complex is a ribosome receptor in the yeast endoplasmic reticulum membrane.     

Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro- alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.     

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).     

Development of secretory activity in the seminal vesicle of the male migratory grasshopper,Melanoplus sanguinipes (fabr.) (Orthoptera : Acrididae)

This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing patterns of its secretion during sexual maturation and after allatectomy in Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). In epithelia from seminal vesicles of newly fledged males, the rough endoplasmic reticulum is well developed, and Golgi complexes are elaborate, which indicates the gland is metabolically active. The cells also contain large glycogen deposits and the lumen microvilli are well differentiated. These ultrastructural features are more dominant in 24-hr-old adults where the cytoplasm is clearly differentiated into basal and apical regions. Basally, the cytoplasm is dominated by rough endoplasmic reticulum, large Golgi complexes, glycogen deposits and numerous mitochondria, while the apical cytoplasm is filled with large secretory and/or lysosomal vesicles. Between days 3 and 7, the ultrastructural features change little other than the rough endoplasmic reticulum cisternae, which become vesicular. Analysis by isoelectric focusing shows that the amount of secretory protein increases with age until day 3, at which time the gland contains its full complement of secretion. In seminal vesicles from allatectomized insects, ultrastructural features of cells and isoelectric focusing patterns of the secretion arc identical to those from normal males.     

Interaction of BiP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex.

Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes. We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane. In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis. In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied. An intact peptide binding pocket is required for this interaction. Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves.     

ATP is required for in vitro assembly of MHC class I antigens but not for transfer of peptides across the ER membrane

We have translated the HLA-B27 heavy chain in vitro and studied its assembly with beta 2-microglobulin and peptide in microsomes from human cells. The assembly process requires ATP. However, the translocation of peptide across the endoplasmic reticulum (ER) membrane does not require ATP, and binding of biotinylated peptide to BiP, an ER luminal protein, occurs after ATP depletion. Proteinase K treatment of the microsomes does not block peptide translocation. Thus, ATP is required in the lumen of the ER for efficient assembly to occur. Microsomes prepared from Raji and T1 cells show similar levels of assembly, whereas assembly in T2 microsomes is 10-fold lower. This difference remains after peptide stimulation of assembly. The inefficient assembly in T2 microsomes is not due to impaired peptide translocation across the ER membrane, as no difference was found compared with microsomes from T1 cells. Instead, the defect seems to reside in the lumen of the ER.     

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.     

Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis

Members of the hsc70 family of molecular chaperones are critical players in the folding and quality control of cellular proteins. Because several human diseases arise from defects in protein folding, the activity of hsc70 chaperones is a potential therapeutic target for these disorders. By using a known hsc70 modulator, 15-deoxyspergualin, as a seed, we identified a novel inhibitor of hsc70 activity. This compound, R/1, inhibits the endogenous and DnaJ-stimulated ATPase activity of hsc70 by 48 and 51%, respectively, and blocks the hsc70-mediated translocation of a preprotein into yeast endoplasmic reticulum-derived microsomal vesicles. Biochemical studies demonstrate that R/1 most likely exerts these effects by altering the oligomeric state of hsc70.     

Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane

The kinetics of the signal recognition particle(SRP)-mediated process of protein translocation across the endoplasmic reticulum membrane was studied by mathematical modeling and complementary experiments. The following results were obtained. (1) A model according to which SRP directs the ribosome, rather than the mRNA, to the membrane is supported by experiments designed to discriminate between the two alternatives. (2) This model describes both steady-state and synchronized translation experiments and makes a number of predictions. (3) The interaction between a nascent protein and SRP may be described by two parameters: (i) a binding constant which can be attributed to the structure of the signal peptide, and (ii) the size of the "SRP-window", i.e. the distance between the first and the last site on the polypeptide chain that can interact with SRP. For preprolactin a binding constant of 1 to 2.5 nmol-1l was estimated. Modeling of the synchronized synthesis of ovalbumin indicates that it has a much weaker binding constant than preprolactin (approximately 0.25 nmol-1l) although we cannot exclude the possibility that the SRP-window may be also smaller. (4) A better understanding of the molecular effects of SRP on translation and translocation through the rough endoplasmic reticulum membrane has been achieved. Inhibition of the steady-state rate of translation by SRP requires a stoichiometric interaction of SRP with ribosomes carrying nascent polypeptide chains and will occur only when ribosomes are piled up back to the initiation site. Translocation, on the other hand, requires only the catalytic action of SRP and is determined by the local concentration of protein-synthesizing ribosomes accumulated at the site(s) of SRP interaction. As a consequence, translational inhibition by SRP may sometimes fail to occur, depending either on the type of protein or on experimental conditions, such as a high mRNA concentration, even if translocation can be demonstrated. (5) A rough extrapolation to the conditions in vivo indicates that all synthesized polypeptide chains destined for translocation across or integration into the endoplasmic reticulum membrane are indeed quantitatively translocated and that no translational inhibition occurs.     

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78

Alterations in Ca(2+) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.     

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.