Knockdown of p180 Eliminates the Terminal Differentiation of a Secretory Cell Line

We have previously reported that the expression in yeast of an integral membrane protein (p180) of the endoplasmic reticulum (ER), isolated for its ability to mediate ribosome binding, is capable of inducing new membrane biogenesis and an increase in secretory capacity. To demonstrate that p180 is necessary and sufficient for terminal differentiation and acquisition of a secretory phenotype in mammalian cells, we studied the differentiation of a secretory cell line where p180 levels had been significantly reduced using RNAi technology and by transiently expressing p180 in nonsecretory cells. A human monocytic (THP-1) cell line, that can acquire macrophage-like properties, failed to proliferate rough ER when p180 levels were lowered. The Golgi compartment and the secretion of apolipoprotein E (Apo E) were dramatically affected in cells expressing reduced p180 levels. On the other hand, expression of p180 in a human embryonic kidney nonsecretory cell line (HEK293) showed a significant increase in proliferation of rough ER membranes and Golgi complexes. The results obtained from knockdown and overexpression experiments demonstrate that p180 is both necessary and sufficient to induce a secretory phenotype in mammalian cells. These findings support a central role for p180 in the terminal differentiation of secretory cells and tissues.     

180-kD ribosome receptor is essential for both ribosome binding and protein translocation

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.     

Alternatively spliced forms of the P180 ribosome receptor differ in their ability to induce the proliferation of rough endoplasmic reticulum

Expression of the canine 180-kDa ribosome receptor p180 in yeast induces the synthesis of RER, and increases the mRNAs of secretory pathway proteins, and protein secretion. To assess whether p180 is a master regulator of cell secretion in mammalian cells, we stably expressed red fluorescent forms of the human p180 variants p180DeltaR (no tandem repeats), p180R (26 repeats), and full-length p180FR (54 repeats) containing different lengths of the tandem repeat ribosome-binding domain in rat pancreatic RINm5F islet beta-cells. All three fluorescent p180 variants localized exclusively to the RER. Cells transfected with p180R were filled with ribosome-studded karmellae, whereas p180DeltaR and p180FR transfectants contained only increased amounts of mostly smooth ER. Unlike in yeast, over-expression of p180R failed to increase the secretory pathway proteins calnexin, SEC61beta, and calreticulin, or ribosome biogenesis. The data suggest that alternative splicing of the p180 tandem repeat domain is a means of regulating the ribosome-binding activity of p180, and potentially the secretory activity of the cell. However, p180 is not a master regulator of mammalian cell secretion as it does not concomitantly trigger the synthesis of protein machinery required to enhance protein synthesis and cell secretion.     

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.     

Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.     

IN02, A Positive Regulator of Lipid Biosynthesis, Is Essential for the Formation of Inducible Membranes in Yeast

Expression of the 180-kDa canine ribosome receptor in Saccharomyces cerevisiae leads to the accumulation of ER-like membranes. Gene expression patterns in strains expressing various forms of p180, each of which gives rise to unique membrane morphologies, were surveyed by microarray analysis. Several genes whose products regulate phospholipid biosynthesis were determined by Northern blotting to be differentially expressed in all strains that undergo membrane proliferation. Of these, the INO2 gene product was found to be essential for formation of p180-inducible membranes. Expression of p180 in ino2Delta cells failed to give rise to the p180-induced membrane proliferation seen in wild-type cells, whereas p180 expression in ino4Delta cells gave rise to membranes indistinguishable from wild type. Thus, Ino2p is required for the formation of p180-induced membranes and, in this case, appears to be functional in the absence of its putative binding partner, Ino4p.     

Functional characterization of the 180-kD ribosome receptor in vivo

A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned and sequenced. The deduced primary structure indicates three distinct domains: an NH2-terminal stretch of 28 uncharged amino acids representing the membrane anchor, a basic region (pI = 10.74) comprising the remainder of the NH2-terminal half and an acidic COOH- terminal half (pI = 4.99). The most striking feature of the amino acid sequence is a 10-amino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem without interruption in the NH2-terminal positively charged region. We postulate that this repeated sequence represents a ribosome binding domain which mediates the interaction between the ribosome and the ER membrane. To substantiate this hypothesis, recombinant full-length ribosome receptor and two truncated versions of this protein, one lacking the potential ribosome binding domain, and one lacking the COOH terminus, were expressed in Saccharomyces cerevisiae. Morphological and biochemical analyses showed all proteins were targeted to, and oriented correctly in the ER membrane. In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or one lacking the COOH-terminal domain were able to bind two to four times as many human ribosomes as control membranes lacking a recombinant protein or microsomes containing a receptor lacking the NH2-terminal basic domain. Electron micrographs of these cells revealed that the expression of all receptor constructs led to a proliferation of perinuclear ER membranes known as "karmellae." Strikingly, in those strains which expressed cDNAs encoding a receptor containing the putative ribosome binding domain, the induced ER membranes (examined in situ) were richly studded with ribosomes. In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding domain were uniformly smooth and free of ribosomes. Cell fractionation and biochemical analyses corroborated the morphological characterization. Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding.     

p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.     

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.     

Homotypic secretory vesicle fusion induced by the protein tyrosine phosphatase MEG2 depends on polyphosphoinositides in T cells

Sec14p homology domains are found in a large number of proteins from plants, yeast, invertebrates, and higher eukaryotes. We report that the N-terminal Sec14p homology domain of the human protein tyrosine phosphatase PTP-MEG2 binds phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in vitro and colocalizes with this lipid on secretory vesicle membranes in intact cells. Point mutations that prevented PtdIns(3,4,5)P(3) binding abrogated the capacity of PTP-MEG2 to induce homotypic secretory vesicle fusion in cells. Inhibition of cellular PtdIns(3,4,5)P(3) synthesis also rapidly reversed the effect of PTP-MEG2 on secretory vesicles. Finally, we show that several different phosphoinositide kinases colocalize with PTP-MEG2, thus allowing for local synthesis of PtdIns(3,4,5)P(3) in secretory vesicle membranes. We suggest that PTP-MEG2 through its Sec14p homology domain couples inositide phosphorylation to tyrosine dephosphorylation and the regulation of intracellular traffic of the secretory pathway in T cells.     

Expansion of the trans-Golgi network following activated collagen secretion is supported by a coiled-coil microtubule-bundling protein, p180, on the ER

A coiled-coil endoplasmic reticulum (ER) protein, p180, was originally reported as a ribosome-binding receptor on the rough ER and is highly expressed in secretory tissues. Recently, we reported new functions of p180 as a microtubule-bundling protein on the ER. Here, we investigated the specific roles of p180 in the Golgi complex organization following stimulated collagen secretion. Targeted depletion of p180 by siRNA transfection caused marked reduction of TGN, while other marker levels for the cis or medial Golgi were not markedly changed. Ascorbate stimulation resulted in trans-Golgi network (TGN) expansion to the periphery in control cells that is characterized by both increased membrane amounts and extended shape. In contrast, loss of p180 resulted in retraction of the TGN regardless of ascorbate stimulation. The TGN developed to the periphery along stabilized microtubule bundles, and overexpression of MTB-1 fragment caused dominant-negative phenotypes. Once disorganized, the retracted TGN did not recover in the absence of p180 despite elevated acetylated tubulin levels. TGN46 and p180 were co-distributed in epithelial basal layer cells of human mucosal and gastrointestinal tissues. Taken together, we propose a novel function of p180-abundant ER on the TGN expansion, both of which are highly developed in various professional secretory cells.     

Subcellular distribution and targeting of the intracellular chloride channel p64.

p64 is an intracellular chloride channel originally identified in bovine kidney microsomes. Using a combination of immunofluorescent and electron microscopic technique, we demonstrate that p64 resides in the limiting membranes of perinuclear dense core vesicles which appear to be regulated secretory vesicles. Heterologous expression of p64 in PancI cells, a cell type which does not normally express p64, results in targeting to a similar compartment. Mutagenesis experiments demonstrate that both the N- and C-terminal domains of the protein independently contribute to subcellular distribution of the protein. The C-terminal domain functions to prevent expression of p64 on the plasma membrane and the N-terminal domain is necessary to deliver p64 to the appropriate membrane compartment.     

In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex

The Sec61p complex forms the core element of the protein translocation complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the beta subunit of the complex (Sec61beta) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61beta cytoplasmic domain (Sec61beta(c)) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61beta(c), like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61beta(c) domain with ribosomal RNA. Like stripped ER membranes, the Sec61beta(c) sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61beta is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61beta, and it has been proposed that Sec61beta assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61beta; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins.     

SHPS-1 induces aggregation of Ba/F3 pro-B cells via an interaction with CD47

SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP) substrate-1), a member of the inhibitory-receptor superfamily that is abundantly expressed in macrophages and neural tissue, appears to regulate intracellular signaling events downstream of receptor protein-tyrosine kinases and integrin-extracellular matrix molecule interactions. To investigate the function of SHPS-1 in a hematopoietic cell line, SHPS-1 was expressed in Ba/F3 cells, an IL-3-dependent pro-B-cell line that lacks endogenous SHPS-1 protein. Interestingly, expression of either SHPS-1, or a mutant lacking the intracellular domain of SHPS-1 (DeltaCT SHPS-1), resulted in the rapid formation of macroscopic Ba/F3 cell aggregates. As the integrin-associated protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we investigated whether CD47 played a role in the aggregation of SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate formation was inhibited by an anti-CD47 Ab. Furthermore, erythrocytes from control, but not from CD47-deficient mice, were able to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes do not express integrins, this result suggested that SHPS-1-CD47 interactions can take place in the absence of a CD47-integrin association. We also present evidence that the amino-terminal Ig domain of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47 binding likely triggers bidirectional intracellular signaling processes, these results demonstrate that this interaction can also mediate cell-cell adhesion.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.     

Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.