The Florey Lecture, 1992. The secretion of proteins by cells.

In eukaryotic cells, protein secretion provides a complex organizational problem. Secretory proteins are first transported, in an unfolded state, across the membrane of the endoplasmic reticulum (ER), and are then carried in small vesicles to the Golgi apparatus and finally to the cell membrane. The ER contains soluble proteins which catalyse the folding of newly synthesized polypeptides. These proteins are sorted from secretory proteins in the Golgi complex: they carry a sorting signal (the tetrapeptide KDEL or a related sequence) that allows them to be selectively retrieved and returned to the ER. This retrieval process also appears to be used by some bacterial toxins to aid their invasion of the cell: these toxins contain KDEL-like sequences and may, in effect, follow the secretory pathway in reverse. The membrane-bound receptor responsible for sorting luminal ER proteins has been identified in yeast by genetic means, and related receptors are found in mammalian cells. Unexpectedly, this receptor has a second role: in yeast it is required to maintain the normal size and function of the Golgi apparatus. By helping to maintain the composition of both ER and Golgi compartments, the KDEL receptor has an important role in the organization of the secretory pathway.     

The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) evokes the ER stress response. The resultant outcomes are cytoprotective but also proapoptotic. ER chaperones and misfolded proteins exit to the secretory pathway and are retrieved to the ER, during which process the KDEL receptor plays a significant role. Using an expression of a mutant KDEL receptor that lacks the ability for ligand recognition, we show that the impairment of retrieval by the KDEL receptor led to a mis-sorting of the immunoglobulin-binding protein BiP, an ER chaperone that has a retrieval signal from the early secretory pathway, which induced intense ER stress response and an increase in susceptibility to ER stress in HeLa cells. Furthermore, we show that the ER stress response accompanied the activation of p38 mitogen-activated protein (MAP) kinases and c-Jun amino-terminal kinases (JNKs) and that the expression of the mutant KDEL receptor suppressed the activation of p38 and JNK1 but not JNK2. The effect of the expression of the mutant KDEL receptor was consistent with the effect of a specific inhibitor for p38 MAP kinases, because the inhibitor sensitized HeLa cells to ER stress. We also found that activation of the KDEL receptor by the ligand induced the phosphorylation of p38 MAP kinases. These results indicate that the KDEL receptor participates in the ER stress response not only by its retrieval ability but also by modulating MAP kinase signaling, which may affect the outcomes of the mammalian ER stress response.     

Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment.

Several soluble proteins that reside in the lumen of the ER contain a specific C-terminal sequence (KDEL) which prevents their secretion. This sequence may be recognized by a receptor that either immobilizes the proteins in the ER, or sorts them from other proteins at a later point in the secretory pathway and returns them to their normal location. To distinguish these possibilities, I have attached an ER retention signal to the lysosomal protein cathepsin D. The oligosaccharide side chains of this protein are normally modified sequentially by two enzymes to form mannose-6-phosphate residues; these enzymes do not act in the ER, but are thought to be located in separate compartments within (or near) the Golgi apparatus. Cathepsin D bearing the ER signal accumulates within the ER, but continues to be modified by the first of the mannose-6-phosphate forming enzymes. Modification is strongly temperature-dependent, which is also a feature of ER-to-Golgi transport. These results support the idea that luminal ER proteins are continuously retrieved from a post-ER compartment, and that this compartment contains N-acetylglucosaminyl-1-phosphotransferase activity.     

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans- Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans- TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL- containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.     

Rescue of a nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutant by cell-penetrating peptides

Mutant membrane proteins are frequently retained in the early secretory pathway by a quality control system, thereby causing disease. An example are mutants of the vasopressin V(2) receptor (V(2)R) leading to nephrogenic diabetes insipidus. Transport-defective V(2)Rs fall into two classes: those retained exclusively in the endoplasmic reticulum (ER) and those reaching post-ER compartments such as the ER/Golgi intermediate compartment. Although numerous chemical or pharmacological chaperones that rescue the transport of ER-retained membrane proteins are known, substances acting specifically in post-ER compartments have not been described as yet. Using the L62P (ER-retained) and Y205C (reaching post-ER compartments) mutants of the V(2)R as a model, we show here that the cell-penetrating peptide penetratin and its synthetic analog KLAL rescue the transport of the Y205C mutant. In contrast, the location of the L62P mutant is not influenced by either peptide because the peptides are unable to enter the ER. We also show data indicating that the peptide-mediated transport rescue is associated with an increase in cytosolic Ca(2+) concentrations. Thus, we describe a new class of substances influencing protein transport specifically in post-ER compartments.     

Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.     

Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD)

Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.     

Intracellular degradation of the transport-impaired human PiZ alpha 1-antitrypsin variant. Biochemical mapping of the degradative event among compartments of the secretory pathway

The naturally occurring PiZ and Pi NullHong Kong variants of the human secretory protein alpha 1-antitrypsin (AAT) are retained within an early compartment of the secretory pathway. Intracellular degradation of these transport-impaired secretory proteins is initiated 30-45 min following their synthesis and translocation into the endoplasmic reticulum (ER). Interestingly, the overall rate of degradation of the retained mutant protein is significantly accelerated when all subcellular compartments are buffered at pH 6. In contrast, degradation is virtually abolished when intravesicular compartments are buffered at pH 8. However, despite this pH sensitivity neither lysosomotrophic amines, leupeptin, or leucine methyl ester have an apparent effect on the intracellular removal of the PiZ variant. The inability of a variety of inhibitors of ER-to-Golgi protein trafficking to hinder the degradative process suggests that degradation of the PiZ variant occurs prior to its delivery to the Golgi complex. To biochemically map the subcellular site of the degradation of the retained mutant protein, a recombinant truncated PiZ variant containing the tetrapeptide KDEL at its carboxyl terminus (a signal for sorting luminal proteins from a post-ER compartment back to the ER) was expressed in cells. Attachment of this ER-recycling signal to the recombinant protein prevented its intracellular degradation. These findings indicate that degradation of the PiZ variant occurs following its export from the ER.     

Sequence of a second human KDEL receptor.

Retention of luminal endoplasmic reticulum (ER) proteins is mediated via a conserved carboxy-terminal tetrapeptide that serves as a signal for their retrieval from subsequent compartments of the secretory pathway. The signal is recognized by a receptor molecule that is believed to cycle between the Golgi apparatus and the ER. This receptor in Saccharomyces cerevisiae is encoded by the ERD2 gene, and a human cDNA homologue of the gene has been isolated. Binding of ligand by the product of this gene results in a shift of its steady-state location from Golgi to ER, suggesting that retrograde transport has been triggered. Here we report the identification of a related human protein with similar properties. This indicates that there are at least two distinct genes in humans that encode functional KDEL receptors.     

Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval.

Dilysine signals confer localization of type I membrane proteins to the endoplasmic reticulum (ER). According to the prevailing model these signals target proteins to the ER by COP I-mediated retrieval from post-ER compartments, whereas the actual retention mechanism in the ER is unknown. We expressed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constructs, which had access to post-ER compartments, the KKAA chimeras were localized to the ER by confocal microscopy and were neither processed by cis-Golgi-specific enzymes in vivo nor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are permanently retained in the ER. The ER localization was nonsaturable and exclusively mediated by the dilysine signal because mutating the lysines to alanines led to cell surface expression of the chimeras. Although the KKAA signal avidly binds COP I in vitro, the ER retention by this signal does not depend on intact COP I in vivo because it was not affected in an epsilon-COP-deficient cell line. We propose that dilysine ER targeting signals can mediate ER retention in addition to retrieval.     

ERGIC-53 KKAA signal mediates endoplasmic reticulum retrieval in yeast

Studies on the ERGIC-53 KKAA signal have revealed a new mechanism for static retention of mammalian proteins in the endoplasmic reticulum (Andersson, H., Kappeler, F., Hauri, H. P. (1999): Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval. J. Biol. Chem. 274,15080 - 15084). To test if this mechanism was conserved in yeast, the ERGIC-53 KKAA signal was transferred on two different yeast reporter proteins. Making use of a genetic assay, we demonstrate that this signal induces COPI-dependent ER retrieval. ER retention of KKAA-tagged proteins was impaired in yeast mutants affected in COPI subunits. Furthermore, biochemical analysis of post-ER carbohydrate modifications detected on reporter proteins indicated that KKAA-tagged proteins recycle continuously within early compartments of the secretory pathway. Therefore in yeast, the KKAA signal might only function as a classical dilysine ER retrieval signal.     

Retrieval from the ER-golgi intermediate compartment is key to the targeting of c-terminally anchored ER-resident proteins

Endoplasmic reticulum (ER) resident proteins may be maintained in the ER by retention, where the leak into post-ER compartments is absent or slow, or retrieval, where a significant leak is countered by retrieval from post-ER compartments. Here the targeting of the C-terminally anchored protein ER-resident protein, cytochrome b5a (cytb5a), considered to be maintained in the ER mainly by the process of retention, is compared with that of sarcolipin (SLN) and phospholamban (PLB); also C-terminally anchored ER-residents. Laser confocal microscopy, and cell fractionation of green fluorescent protein-tagged constructs expressed in COS 7 cells indicate that while calnexin appears to be retained in the ER with no evidence of leak into the ER-Golgi intermediate compartment (ERGIC), significant amounts of cytb5a, SLN, and PLB are detectable in the ERGIC, indicating that there is considerable leak from the ER. This is supported by an in vitro budding assay that shows that while small amounts of calnexin appear in the transport vesicles budding off from the ER, significant amounts of cytb5a and SLN are found in such vesicles. These data support the hypothesis that retrieval plays a major role in ensuring that C-terminally anchored proteins are maintained in the ER.     

Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum.

Resident luminal endoplasmic reticulum (ER) proteins carry a targeting signal (usually KDEL in animal cells) that allows their retrieval from later stages of the secretory pathway. In yeast, the receptor that promotes this selective retrograde transport has been identified as the product of the ERD2 gene. We describe here the properties of a human homolog of this protein (hERD2). Overproduction of hERD2 improves retention of a protein with a weakly recognized variant signal (DDEL). Moreover, overexpression of KDEL or DDEL ligands causes a redistribution of hERD2 from the Golgi apparatus to the ER. Mutation of hERD2 alters the ligand specificity of this effect, implying that it interacts directly with the retained proteins. Ligand control of receptor movement may limit retrograde flow and thus minimize fruitless recycling of secretory proteins.     

The retention signal for soluble proteins of the endoplasmic reticulum

The lumen of the endoplasmic reticulum (ER) contains a number of soluble proteins, many of which help the maturation of newly synthesized secretory proteins. Retention of these resident proteins in the ER is dependent on a carboxy-terminal signal, which in animal cells is usually Lys-Asp-Glu-Leu (KDEL). This signal is thought to be recognized by a membrane-bound receptor that continually retrieves the proteins from a later compartment of the secretory pathway and returns them to the ER.     

Different sorting of Lys-Asp-Glu-Leu proteins in rat liver.

Most of the resident soluble proteins of the endoplasmic reticulum (ER) seem to be sorted into this compartment via their COOH-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). This sorting is supposed to occur in a post-ER compartment. Three resident soluble ER glycoproteins belonging to the KDEL family are CaBP1, CaBP2, CaBP3 (= calreticulin), and CaBP4 (= grp94) (Nguyen Van, P., Peter, F., and S?ling, H.-D. (1989) J. Biol. Chem. 264, 17494-17501). In rat liver, calreticulin possesses a carbohydrate moiety of the complex hybrid type with terminal galactoses (Nguyen Van, P., Peter, F., and S?ling, H.-D. (1989) J. Biol. Chem. 264, 17494-17501). We can show now that practically all calreticulin molecules (and not only a fraction) possess terminal galactoses as well as the COOH-terminal KDEL sequence. This as well as pulse-chase experiments performed at 37 and 15 degrees C indicate that calreticulin must have passed through the trans-Golgi. Subcellular fractionations of post-mitochondrial supernatants from isolated rat hepatocytes by sucrose-Nycodenz gradient centrifugation revealed that calreticulin is confined mainly to the rough ER, grp94 mainly to the smooth ER. CaBP1, a member of the thioredoxin family, was recovered in fractions which most likely represent the intermediate compartment. This indicates that KDEL is a sorting signal which leads to the retention of these proteins in the pre-Golgi compartments. However, additional factors, most likely residing within the specific KDEL protein itself, determine the final location of the protein within the pre-Golgi compartments. This is underlined by experiments in which the density dependent distribution of total KDEL proteins was studied using a COOH-terminal KDEL-specific antibody.     

A molecular specificity code for the three mammalian KDEL receptors

AC-terminal KDEL-like motif prevents secretion of soluble endoplasmic reticulum (ER)–resident proteins. This motif interacts with KDEL receptors localized in the intermediate compartment and Golgi apparatus. Such binding triggers retrieval back to the ER via a coat protein I–dependent pathway. To date, two human KDEL receptors have been reported. Here, we report the Golgi localization of a third human KDEL receptor. Using a reporter construct system from a screen of 152 variants, we identified 35 KDEL-like variants that result in efficient ER localization but do not match the current Prosite motif for ER localization ([KRHQSA]-[DENQ]-E-L). We cloned 16 human proteins with one of these motifs and all were found in the ER. A subsequent screen by bimolecular fluorescence complementation determined the specificities of the three human KDEL receptors. Each KDEL receptor has a unique pattern of motifs with which it interacts. This suggests a specificity in the retrieval of human proteins that contain different KDEL variants.     

E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells.

The E5 oncoprotein encoded by bovine papillomavirus type 1 is a homodimeric, hydrophobic polypeptide which is localized predominantly in Golgi membranes and which transforms several cell types apparently by inducing tyrosine phosphorylation of the platelet-derived growth factor receptor (PDGF-R). While the precise mechanism of receptor activation is unknown, E5 associates with several cellular proteins, including PDGF-R and the 16K V-ATPase protein, and induces the preferential phosphorylation of immature, Endo H-sensitive forms of the receptor. To evaluate whether E5 accumulation in the Golgi was requisite for receptor phosphorylation and cell transformation, we sequestered the E5 protein in the endoplasmic reticulum (ER)/cis Golgi by appending the ER retention KDEL sequence to its C-terminus. In transient assays and in cell lines, E5/KDEL protein and E5/KDEL* protein (a defective variant of KDEL), were stable and formed homodimers normally. E5/KDEL*, similar to wt E5, localized to the Golgi and was transformation-proficient. In contrast, E5/KDEL failed to concentrate in the Golgi and was transformation-incompetent. Despite these critical defects, however, E5/KDEL formed stable complexes with immature PDGF-R and 16K and, even more unexpectedly, induced the phosphorylation of both mature and immature PDGF-R on tyrosine residues to the same level as wt E5. These data demonstrate that E5 can bind and induce PDGF-R phosphorylation in the ER/cis Golgi, but that successful mitogenic signalling (and consequent cell transformation) requires the translocation of E5/receptor complexes to distal Golgi compartments.     

Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia

During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.     

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.