Cutting edge: Tapasin is retained in the endoplasmic reticulum by dynamic clustering and exclusion from endoplasmic reticulum exit sites

Tapasin retains empty or suboptimally loaded MHC class I molecules in the endoplasmic reticulum (ER). However, the molecular mechanism of this process and how tapasin itself is retained in the ER are unknown. These questions were addressed by tagging tapasin with the cyan fluorescent protein or yellow fluorescent protein (YFP) and probing the distribution and mobility of the tagged proteins. YFP-tapasin molecules were functional and could be isolated in association with TAP, as reported for native tapasin. YFP-tapasin was excluded from ER exit sites even after accumulation of secretory cargo due to disrupted anterograde traffic. Almost all tapasin molecules were clustered, and these clusters diffused freely in the ER. Tapasin oligomers appear to be retained by the failure of the export machinery to recognize them as cargo.     

Cell transformation potential of a HER2 transmembrane domain deletion mutant retained in the endoplasmic reticulum.

The HER2 (c-erbB-2) gene encodes a protein, p185HER2, which possesses all of the structural characteristics and functional properties of a growth factor receptor, although its ligand has not yet been well characterized. HER2 is the human homolog of the rat proto-oncogene neu and is closely related to the gene encoding the epidermal growth factor receptor. Amplification of this gene and overexpression have been found to be a prognostic criterion for a 30% subpopulation of human breast cancer patients. In this study, we investigated the role of the transmembrane-spanning sequence in the biosynthesis and localization of p185HER2. A truncation mutant lacking the cytoplasmic and transmembrane domains was glycosylated and efficiently secreted. However, a mutant lacking only the transmembrane-spanning sequence was incompletely glycosylated and failed to reach the cell surface. Unexpectedly, although this deletion mutant was retained in the endoplasmic reticulum membrane, it was still able to transform NIH 3T3 cells when expressed at high levels.     

Mutant prion proteins are partially retained in the endoplasmic reticulum

Familial prion diseases are linked to point and insertional mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP to the infectious isoform. In this report, we have investigated the subcellular localization of PrP molecules carrying pathogenic mutations using immunofluorescence staining, immunogold labeling, and PrP-green fluorescent protein chimeras. To facilitate visualization of the mutant proteins, we have utilized a novel Sindbis viral replicon engineered to produce high protein levels without cytopathology. We demonstrate that several different pathogenic mutations have a common effect on the trafficking of PrP, impairing delivery of the molecules to the cell surface and causing a portion of them to accumulate in the endoplasmic reticulum. These observations suggest that protein quality control in the endoplasmic reticulum may play an important role in prion diseases, as it does in some other inherited human disorders. Our experiments also show that chimeric PrP molecules with the sequence of green fluorescent protein inserted adjacent to the glycolipidation site are post-translationally modified and localized normally, thus documenting the utility of these constructs in cell biological studies of PrP.     

Acetylcholine receptor in a C2 muscle cell variant is retained in the endoplasmic reticulum

We have examined the properties and intracellular localization of acetylcholine receptors in the C2 muscle cell line and in a variant (T-) that accumulates AChR intracellularly. On immunoblots, the subunit structures of the AChR from wild-type and T- cells were similar except that the gamma and delta subunits of the variant AChR had altered mobilities. Digestion with endoglycosidases H and F demonstrated that this difference results from a failure of high-mannose N-linked oligosaccharides on AChR subunits to be processed to complex forms in the variant. N-linked glycosylation of other proteins in the variant was normal. When examined by immunocytochemistry, the distribution of internal AChR in wild-type cells was consistent with a location both in the endoplasmic reticulum and in the Golgi. Variant cells, however, showed no evidence of Golgi staining. Subcellular fractionation experiments also demonstrated AChR in the Golgi fractions of wild-type cells, but not in those derived from T- cells. We conclude that in T- myotubes most of the AChR fails to be transported out of the endoplasmic reticulum.     

Aberrant quality control in the endoplasmic reticulum impairs the biosynthesis of pulmonary surfactant in mice expressing mutant BiP

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which alleviates protein overload in the secretory pathway. Although the UPR is activated under diverse pathological conditions, its physiological role during development and in adulthood has not been fully elucidated. Binding immunoglobulin protein (BiP) is an ER chaperone, which is central to ER function. We produced knock-in mice expressing a mutant BiP lacking the retrieval sequence to cause a defect in ER function without completely eliminating BiP. In embryonic fibroblasts, the UPR compensated for mutation of BiP. However, neonates expressing mutant BiP suffered respiratory failure due to impaired secretion of pulmonary surfactant by alveolar type II epithelial cells. Expression of surfactant protein (SP)-C was reduced and the lamellar body was malformed, indicating that BiP plays a critical role in the biosynthesis of pulmonary surfactant. Because pulmonary surfactant requires extensive post-translational processing in the secretory pathway, these findings suggest that in secretory cells, such as alveolar type II cells, the UPR is essential for managing the normal physiological ER protein overload that occurs during development. Moreover, failure of this adaptive mechanism may increase pulmonary susceptibility to environmental insults, such as hypoxia and ischemia, ultimately leading to neonatal respiratory failure.     

Morphology and function of the endoplasmic reticulum

Comparison with the findings in the cells of other plants and animals showed that the endoplasmic reticulum in the root apex ofFagopyrum has the same general character and function as in other biological objects, i.e. in secretory processes and especially in this case in the transport of the substances produced. Detailed studies of the morphology and activity of the endoplasmic reticulum showed some functional differences which are characteristic for this object. The endoplasmic reticulum participates apparently in the transport of the mass of known but functionally and nomenclatorically controversial formations which here are called dense bodies. Dense bodies exist inFagopyrum in a considerable amount as compared to other objects. Frequent contact of the dense bodies with the ends of the endoplasmic reticulum, contact with the endoplasmic retieulum passing through the plasmodesm, accumulation of the dense bodies along the cell wall and in proximal distance of the plasmodesms and intensive staining of some plasmodesms was observed. The dense vacuoles in this object represent dilated spaces of the endoplasmic reticulum which apparently have the function of reservoirs of the dense mass. The endoplasmic reticulum in the calyptra cells appears to participate in the formation of the cell walls. This object differs hereby from others, where the participation of the Golgi apparatus has been observed in this function.     

A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37 degrees C but exits the ER and accumulates in endosomal structures in cells grown at 31 degrees C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37 degrees C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37 degrees C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.     

Pannexin channels in ATP release and beyond: An unexpected rendezvous at the endoplasmic reticulum

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²⁺, for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²⁺-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²⁺ leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²⁺ homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²⁺ signalling.     

CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor.

We analyzed coexpression of the human immunodeficiency virus type 1 glycoprotein precursor, gp160, and its cellular receptor CD4 in HeLa cells to determine whether the two molecules can interact prior to transport to the cell surface. Results of studies employing coprecipitation, analysis of oligosaccharide processing, and immunocytochemistry showed that newly synthesized CD4 and gp160 form a complex prior to transport from the endoplasmic reticulum (ER). CD4 expressed by itself was transported efficiently from the ER to the cell surface, but the complex of CD4 and gp160 was retained in the ER. This retention of CD4 within the ER is probably a consequence of the very inefficient transport of gp160 itself (R. L. Willey, J. S. Bonifacino, B. J. Potts, M. A. Martin, and R. D. Klausner, Proc. Natl. Acad. Sci. USA 85:9580-9584, 1988). Retention of CD4 in the ER by gp160 may partially explain the down regulation of CD4 in human immunodeficiency virus type 1-infected T cells. Inhibition of CD4 transport appears to be a consequence of the interaction of two membrane-bound molecules, because a complex of CD4 and gp120 (the soluble extracellular domain of gp160) was transported rapidly and efficiently from the ER.     

Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immunohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies.     

Endogenous meltrin alpha is ubiquitously expressed and associated with the plasma membrane but exogenous meltrin alpha is retained in the endoplasmic reticulum

Meltrin alpha (ADAM12) is a member of the ADAM (MDC) protein family characterized by the presence of metalloprotease and disintegrin domains. ADAM proteins contain single transmembrane domains, and the processed mature proteins are postulated to span the plasma membrane. It has been reported that transfection of a truncated meltrin alpha cDNA lacking the prodomain and metalloprotease domain promotes skeletal muscle cell fusion. We show here that meltrin alpha was constitutively expressed in both undifferentiated and differentiated C2 skeletal muscle cells and also in fibroblasts. Both its precursor and processed mature forms were present in these cells. Thus, meltrin alpha may play general roles in addition to its roles in myogenesis. Since endogenous meltrin alpha cannot be detected by immunofluorescence microscopy, we examined the location of the exogenously expressed protein by transfection. Unexpectedly, the exogenously expressed meltrin alpha was located to a network structure of the endoplasmic reticulum (ER) but not to the plasma membrane. Cell fractionation revealed that the intrinsic mature protein was associated with the plasma membrane. However, the exogenously expressed protein remained unprocessed. These results seem to imply that the exogenously expressed meltrin alpha is not translocated from the ER to the trans-Golgi network, where a processing enzyme resides, and that it is consequently not converted to the mature form. Thus, the transfected meltrin alpha is unlikely to exert its physiological functions. Conversely, the ER may serve as a reservoir of the latent form of intrinsic meltrin alpha.     

Endocytosis of simian virus 40 into the endoplasmic reticulum

The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.     

An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum

Arabidopsis contains 34 genes that are predicted to encode calcium-dependent protein kinases (CDPKs). CDPK enzymatic activity previously has been detected in many locations in plant cells, including the cytosol, the cytoskeleton, and the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. We investigated the subcellular location of one Arabidopsis CDPK, AtCPK2, in detail. Membrane-associated AtCPK2 did not partition with the plasma membrane in a two-phase system. Sucrose gradient fractionation of microsomes demonstrated that AtCPK2 was associated with the endoplasmic reticulum (ER). AtCPK2 does not contain transmembrane domains or known ER-targeting signals, but does have predicted amino-terminal acylation sites. AtCPK2 was myristoylated in a cell-free extract and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In plants, the G2A mutation decreased AtCPK2 membrane association by approximately 50%. A recombinant protein, consisting of the first 10 amino acids of AtCPK2 fused to the amino-terminus of beta-glucuronidase, was also targeted to the ER, indicating that the amino terminus of AtCPK2 can specify ER localization of a soluble protein. These results indicate that AtCPK2 is localized to the ER, that myristoylation is likely to be involved in the membrane association of AtCPK2, and that the amino terminal region of AtCPK2 is sufficient for correct membrane targeting.     

CPEB4 is a cell survival protein retained in the nucleus upon ischemia or endoplasmic reticulum calcium depletion

The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.     

A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum

The major physiological role of the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) is to protect elastic fibers in the lung from excessive hydrolysis by neutrophil elastase. Genetic deficiency of alpha 1-AT predisposes individuals toward the development of emphysema. We have cloned and characterized a mutant alpha 1-AT gene from an individual exhibiting a total absence of immunoreactive alpha 1-AT in serum. Nucleotide sequence analysis of this "null" allele has demonstrated a TC dinucleotide deletion within the codon for Leu318 in exon IV. This frame-shift mutation results in the generation of a premature termination codon at residue 334, which is upstream of the active inhibitory site. To determine the biochemical basis of the null phenotype, the mutant and normal genes were transferred into mouse hepatoma cells for expression analysis. Pulse-chase experiments demonstrated that the mutant gene is expressed into a truncated protein of 45 kDa, which is retained within the rough endoplasmic reticulum. The complete lack of secretion of the truncated protein is consistent with the absence of immunoreactive alpha 1-AT in the patient's serum. In addition, a G to A transition was identified in exon II of the mutant gene, changing the codon for Arg101 to His101. Finally, an A to C transversion was identified in exon V changing the codon for Glu376 to Asp376. Since the latter conservative amino acid substitution has previously been identified in the common PiM2 variant, the frame-shift mutation might have occurred on a PiM2 background chromosome. Using the birthplace of this index case, this mutant alpha 1-AT allele has been designated "nullHong Kong."     

Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.     

The role of connexin40 in developing atrial conduction

Connexin40 (Cx40) is the main connexin expressed in the murine atria and ventricular conduction system. We assess here the developmental role of Cx40 in atrial conduction of the mouse. Cx40 deficiency significantly prolonged activation times in embryonic day 10.5, 12.5 and 14.5 atria during spontaneous activation; the severity decreased with increasing age. In a majority of Cx40 deficient mice the impulse originated from an ectopic focus in the right atrial appendage; in such a case the activation time was even longer due to prolonged activation. Cx40 has thus an important physiological role in the developing atria.     

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants

Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.     

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.     

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.