Variants of the carboxyl-terminal KDEL sequence direct intracellular retention

Soluble proteins which reside in the lumen of the endoplasmic reticulum share a common carboxyl-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). Addition of the tetrapeptide to a normally secreted protein is both necessary and sufficient to cause retention in the endoplasmic reticulum. In order to characterize the critical residues in the KDEL signal, cDNAs encoding proneuropeptide Y (pro-NPY) with the 4-amino acid carboxyl-terminal extension KDEL or a series of KDEL variants were expressed in the AtT-20 cell line. AtT-20 cells, a mouse anterior pituitary corticotrope cell line, synthesize, process, and secrete the pro-ACTH/endorphin precursor. Since post-translational processing in AtT-20 cells has been extensively characterized, it provides a model system in which the processing of a foreign peptide precursor (pro-NPY) and the endogenous precursor (pro-ACTH/endorphin) can be compared. Altered cDNAs encoding pro-NPY with KDEL, DKEL, RDEL, KNEL, KDQL, or KDEA at the COOH terminus were used to generate stable AtT-20 cell lines. The processing of pro-NPY to neuropeptide Y and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic peptide mapping, and radiosequencing. Addition of the tetrapeptides KDEL, DKEL, RDEL, or KNEL to the COOH terminus of the neuropeptide Y precursor, a peptide hormone normally processed and secreted from neuronal cells, caused complete intracellular retention of the unprocessed prohormone in AtT-20 cells. However, KDQL and KDEA-extended pro-NPY molecules were processed and secreted like wild-type pro-NPY when expressed in AtT-20 cells. The secretion of proNPY-derived peptides in these cell lines paralleled secretion of endogenous pro-ACTH/endorphin-derived products under both basal and stimulated conditions. These mutagenesis studies demonstrate that variants of the KDEL retention signal can direct intracellular retention.     

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.     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.     

Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin

We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y. (2001) J. Biol. Chem. 276, 6867-6873). In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin. The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.     

Retention of a type II surface membrane protein in the endoplasmic reticulum by the Lys-Asp-Glu-Leu sequence.

Soluble luminal proteins of the endoplasmic reticulum (ER) are known to be retained by a tetrapeptide retention signal, KDEL. We report in this communication that the KDEL sequence when appended to the carboxy terminus of a cell surface membrane protein, dipeptidyl peptidase IV (DPPIV), resulted in its retention in the endoplasmic reticulum of transfected Madin-Darby canine kidney cells as assessed by indirect immunofluorescence. Selective surface biotinylation revealed that about 90-95% of the expressed DPPIV was retained in the ER. Appendance of the sequence KDEV did not, however, result in ER retention, illustrating the functional specificity of the retention signal. The ER retention was not due to misfolding of the mutant protein, as the mutant proteins remained enzymatically active. Our data suggest that the KDEL receptor is able to recognize and recycle type II membrane proteins containing a carboxyl-terminal KDEL sequence and postulates the existence of such yet to be identified endogenous proteins.     

A different intracellular distribution of a single reporter protein is determined at steady state by KKXX or KDEL retrieval signals

To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.     

Analysis of the retention signals of two resident luminal endoplasmic reticulum proteins by in vitro mutagenesis

Protein disulfide isomerase (PDI, ERp59), ERp72, and ERp61 are luminal proteins of the endoplasmic reticulum (ER) that are characterized by the presence of sequences corresponding to the active site regions of PDI. Each one of these proteins possesses a different COOH-terminal tetrapeptide ER retention signal. In order to investigate what other tetrapeptide sequences could serve as retention signals and to determine to what extent the function of the retention signal is modulated by the protein carrying the signal, we have constructed a set of mutants of two of these resident ER proteins, PDI and ERp72. In each of these proteins, the wild type tetrapeptide sequences were replaced by each member of the set of the 12 possible combinations consisting of (K,R,Q)-(D,E)-(D,E)-L. Analysis of the efficiency of retention of the variant proteins when each was transiently expressed in COS cells showed that the retention efficiencies vary with both the COOH-terminal sequence and with the protein that carries this sequence.     

Soluble aggregates of the human PiZ alpha 1-antitrypsin variant are degraded within the endoplasmic reticulum by a mechanism sensitive to inhibitors of protein synthesis.

Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within transfected mouse hepatoma cells and is subjected to intracellular degradation (Le, A., Graham, K., and Sifers, R.N. (1990) J. Biol. Chem. 265, 14001-14007). The retained protein undergoes a discrete size reduction that results from the modification of its endoglycosidase H-sensitive oligosaccharides and is inhibited by 1-deoxymannojirimycin. Metabolic poisons and inhibitors of protein synthesis perturb the intracellular degradation of the retained protein but do not affect its size reduction. The ability of metabolic poisons to influence the degradation of the PiZ variant in cells treated with brefeldin A indicates that export of the macromolecule from the endoplasmic reticulum (ER) is not the energy-dependent component of its degradation. Subcellular fractionation experiments have verified that both the size reduction and degradation of the retained PiZ variant occur within the rough ER. Finally, sedimentation velocity centrifugation analysis of radiolabeled cell extracts has indicated that approximately 80% of the PiZ variant consists as soluble aggregates immediately after its synthesis. An inability to detect more extensive aggregation during the retention period supports our previous conclusion that only a small fraction of the macromolecules actually form large insoluble aggregates (Graham, K.S., Le, A., and Sifers, R.N. (1990) J. Biol. Chem. 265, 20463-20468). Overall, these findings indicate that soluble aggregates of the PiZ variant are degraded within the ER by a mechanism sensitive to inhibitors of protein synthesis.     

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.     

Secretion of alpha-1-proteinase inhibitor requires an almost full length molecule.

In the human disease alpha-1-proteinase inhibitor deficiency, some variants of human alpha-1-proteinase inhibitor are not secreted. These secretory variants contain frameshift mutations leading to products with normal amino acid sequences to the points of the mutations followed by short, aberrant C-terminal sequences and then premature termination (Nukiwa, T., Takahashi, H., Brantly, M., Courtney, M., and Crystal, R. (1987) J. Biol. Chem. 262, 11999-12004; Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335; Curiel, D., Brantly, M., Curiel, E., Stier, L., and Crystal, R. G. (1989) J. Clin. Invest. 83, 1144-1152). To examine possible causes for lack of secretion of these null variants, we have altered the alpha-1-proteinase inhibitor cDNA to encode a series of abbreviated forms of this protein that retain authentic sequences to the points of truncation. Examination of the fates of these shortened proteins in transiently transfected Cos 1 cells indicates that the aberrant C-terminal sequences in the naturally occurring variants are not responsible for their lack of secretion and show that truncation prior to Pro391 prevents movement from the endoplasmic reticulum to the Golgi apparatus and therefore secretion. These truncated forms of alpha-1-proteinase inhibitor do not form inclusion bodies in the endoplasmic reticulum, rather they are degraded, probably by the pre-Golgi pathway. Our results support the idea that a sequence of at least 391 of the normal 394 residues is essential for the secretion of alpha-1-proteinase inhibitor and suggest that residue 391 plays an especially important role, perhaps in allowing or directing proper folding or as part of a transport signal, in the secretion of this protein.     

The carboxylesterase family exhibits C-terminal sequence diversity reflecting the presence or absence of endoplasmic-reticulum-retention sequences.

Resident proteins of the endoplasmic reticulum lumen are continuously retrieved from an early Golgi compartment by a receptor-mediated mechanism. The sorting or retention sequence on the endoplasmic reticulum proteins is located at the C-terminus and was initially shown to be the tetrapeptide KDEL in mammalian cells and HDEL in Saccharomyces cerevisiae. The carboxylesterases are a large family of enzymes primarily localized to the lumen of the endoplasmic reticulum. Retention sequences in these proteins have been difficult to identify due to atypical and heterogeneous C-terminal sequences. Utilizing the polymerase chain reaction with degenerate primers, we have identified and characterized the C-termini of four members of the carboxylesterase family from rat liver. Three of the carboxylesterases sequences contained C-terminal sequences (HVEL, HNEL or HTEL) resembling the yeast sorting signal which were reported to be non-functional in mammalian cells. A fourth carboxylesterase contained a distinct C-terminal sequence, TEHT. A full-length esterase cDNA clone, terminating in the sequence HVEL, was isolated and was used to assess the retention capabilities of the various esterase C-terminal sequences. This esterase was retained in COS-1 cells, but was secreted when its C-terminal tetrapeptide, HVEL, was deleted. Addition of C-terminal sequences containing HNEL and HTEL resulted in efficient retention. However, the C-terminal sequence containing TEHT was not a functional retention signal. Both HDEL, the authentic yeast retention signal, and KDEL were efficient retention sequences for the esterase. These studies show that some members of the rat liver carboxylesterase family contain novel C-terminal retention sequences that resemble the yeast signal. At least one member of the family does not contain a C-terminal retention signal and probably represents a secretory form.     

Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide

Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical protomers linked by a four-stranded antiparallel beta-sheet consisting of the NH2- and COOH-terminal segments (Weber, I.T. (1990) J. Biol. Chem. 265, 10492-10496). Kinetic analysis of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA buffer, 37 degrees C, the equilibrium dissociation constant, Kd = 3.6 +/- 1.9 nM. We found that the tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic analysis shows that the inhibitor binds to the inactive protomers and prevents their association into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equilibrium experiments in which the apparent molecular weight of the enzyme was observed to be 20,800 +/- 1,500 and 12,100 +/- 300, in the absence and presence of the COOH-terminal tetrapeptide, respectively. The dissociation constant of the protomer-inhibitor complex is Ki = 45.1 +/- 1.8 microM. This is the first kinetic analysis and direct experimental demonstration of noncovalent dissociative inhibition.     

Membrane recruitment of coatomer and binding to dilysine signals are separate events

It has previously been shown that transport of newly synthesized proteins and the structure of the Golgi complex are affected in the Chinese hamster ovary cell line ldlF, which bears a temperature-sensitive mutation in the Coat protein I (COPI) subunit epsilon-COP (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224; Hobbie, L., Fisher, A. S., Lee, S., Flint, A., and Krieger, M. (1994) J. Biol. Chem. 269, 20958-20970). Here, we pinpoint the site of the secretory block to an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi complex and show that the distributions of ER-Golgi recycling proteins, such as KDEL receptor and p23, as well as resident Golgi proteins, such as mannosidase II, are accordingly affected. At the nonpermissive temperature, neither the stability of the COPI complex nor its recruitment to donor Golgi membranes is affected. However, the binding of coatomer to the dilysine-based ER-retrieval motif is impaired in the absence of epsilon-COP, suggesting that dilysine signal binding is not the major means of COPI recruitment. Because expression of the exogenous chimera of epsilon-COP and green fluorescent protein in ldlF cells at nonpermissive temperature rapidly restores the wild type properties, epsilon-COP is likely to play an important role in the cargo selection events mediated by COPI.     

Cab45, a novel (Ca2+)-binding protein localized to the Golgi lumen

We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca2+ due to the presence of six EF-hand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol Chem. 269:19142- 19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi. Cab45 is the first calcium- binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment.     

Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain

Recently, we fused a matrix-targeting signal to a large fragment of vesicular stomatitis virus G protein, which contains near its COOH-terminus a well-characterized endoplasmic reticulum (ER) stop-transfer sequence; the hybrid G protein was sorted to the inner mitochondrial membrane (Nguyen, M., and G. C. Shore. 1987. J. Biol. Chem. 262:3929-3931). Here, we show that the 19 amino acid G stop-transfer domain functions in an identical fashion when inserted toward the COOH-terminus of an otherwise normal matrix precursor protein, pre-ornithine carbamyl transferase; after import, the mutant protein was found anchored in the inner membrane via the stop-transfer sequence, with its NH2 terminus facing the matrix and its short COOH-terminal tail located in the intermembrane space. However, when the G stop-transfer sequence was placed near the NH2 terminus, the protein was inserted into the outer membrane, in the reverse orientation (NH2 terminus facing out, with a large COOH-terminal fragment located in the intermembrane space). These observations for mitochondrial topogenesis can be explained by a simple extension of existing models for ER sorting.     

Accumulation of the insoluble PiZ variant of human alpha 1-antitrypsin within the hepatic endoplasmic reticulum does not elevate the steady-state level of grp78/BiP

Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within cells and subsequently degraded within a pre-Golgi nonlysosomal compartment that is apparently separate from the endoplasmic reticulum (ER) (Le, A., Graham, K. S., and Sifers, R. N. (1990) J. Biol. Chem. 265, 14001-14007). Despite this phenomenon, human patients and PiZ-bearing transgenic mice exhibit an accumulation of the undegraded protein as insoluble aggregates within distended cisternae of the hepatic ER (Carlson, J. A., Rogers, B. B., Sifers, R. N., Finegold, M. J., Clift, S. M., DeMayo, F. J., Bullock, D. W., and Woo, S. L. C. (1989) J. Clin. Invest. 83, 1183-1190). Immunoprecipitation of the PiZ variant from pulse-radiolabeled hepatocytes from the transgenic animals has demonstrated that a minute quantity of the newly synthesized mutant protein is apparently resistant to degradation and accumulates gradually within the particulate fraction of the cell. Although the steady-state level of the resident ER protein grp78/BiP is elevated in response to the accumulation of malfolded proteins within that subcellular compartment, this phenomenon is not elicited by the accumulation of the insoluble PiZ variant. These results indicate that neither the accumulation of this malfolded protein within the ER nor even the distention of that subcellular compartment is sufficient to cause the up-regulation of grp78/BiP levels. The interpretation of these results with regard to the factors that regulate the levels of grp78/BiP in the ER is discussed.     

Protein disulfide-isomerase in rat exocrine pancreatic cells is exported from the endoplasmic reticulum despite possessing the retention signal

Recently we found by immunogold electron microscopy that protein disulfide-isomerase (PDI), a major resident protein in the lumen of the endoplasmic reticulum (ER) of many cells, is exceptionally localized in rat exocrine pancreatic cells not only in the ER but also in plasma membranes and other organelles along secretory pathway (Akagi, S., Yamamoto, A., Yoshimori, T., Masaki, R., Ogawa, R., and Tashiro, Y. (1988) J. Histochem. Cytochem. 36, 1069-1074). These observations suggest that another type of PDI, e.g. one with a defective ER retention signal, might exist and be transported in the exocrine pancreatic cells. We therefore compared biochemical and immunochemical properties of the transported PDI with the authentic ER resident PDI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, urea-polyacrylamide gel electrophoresis, and isoelectric focusing showed that the former was indistinguishable from the latter. We prepared a polyclonal antibody against the synthetic hexapeptide, which corresponds to the carboxyl terminus of PDI containing the putative ER retention signal "KDEL." The epitopes of this antibody (anti-KDEL antibody) were located within the KDEL sequence. Anti-KDEL antibody reacted with PDI in both the plasma membranes and the ER of rat pancreatic cells in immunoblot analysis as well as in immunogold electron microscopy. These results suggest that PDI exported from the ER to the plasma membranes in rat exocrine pancreatic cells possesses the KDEL sequence.     

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).     

Molecular cloning and nucleotide sequence of cDNA of microsomal carboxyesterase E1 of rat liver

cDNA clones of the mRNA for rat liver carboxyesterase E1, one of the carboxyesterases exclusively located on the luminal side of microsomal vesicles, were isolated. Sequence analysis of 2 kbp long cDNA revealed the primary structure of carboxyesterase E1, which consisted of 549 amino acids (Mr 60, 171.71) and contained an extra peptide of 18 amino acids at the NH2-terminus of the mature enzyme. Comparison of the deduced primary structure and sequences of some proteolytic fragments of the purified enzyme indicated the multiplicity of the enzyme. The extra peptide at the NH2-terminal had features in common with the signal peptides of most secretory proteins. However, no polar amino acid residues existed before the hydrophobic core of the signal peptide. A new interpretation is proposed to explain how the signal peptide without the NH2-terminal polar residues works. A tetrapeptide (KDEL) which was shown to keep a few microsomal proteins in the lumen of the endoplasmic reticulum was not found in the primary structure of carboxyesterase E1, which suggested the existence of another mechanism for retention of proteins in the lumen of endoplasmic reticulum. Carboxyesterase E1 showed significant homology with the COOH-terminal portion of thyroglobulin.