JPDI,a novel endoplasmic reticulum-resident protein containing both a BiP-interacting J-domain and thioredoxin-like motifs

Several endoplasmic reticulum (ER)-resident luminal proteins have a characteristic ER retrieval signal, KDEL, or its variants at their C terminus. Our previous work searching EST databases for proteins containing the C-terminal KDEL motif predicted some novel murine proteins, one of which designated JPDI (J-domain-containing protein disulfide isomerase-like protein) is characterized in this study. The primary structure of JPDI is unique, because in addition to a J-domain motif adjacent to the N-terminal translocation signal sequence, four thioredoxin-like motifs were found in a single polypeptide. As examined by Northern blotting, the expression of JPDI was essentially ubiquitous in tissues and almost independent of ER stress. A computational prediction that JPDI is an ER-resident luminal protein was experimentally supported by immunofluorescent staining of epitope-tagged JPDI-expressing cells together with glycosylation and protease protection studies of this protein. JPDI probably acts as a DnaJ-like partner of BiP, because a recombinant protein carrying the J-domain of JPDI associated with BiP in an ATP-dependent manner and enhanced its ATPase activity. We speculate that for the folding of some proteins in the ER, chaperoning by BiP and formation of proper disulfide bonds may synchronously occur in a JPDI-dependent manner.     

Interaction of Autographa californica multiple nucleopolyhedrovirus cathepsin protease progenitor (proV-CATH) with insect baculovirus chitinase as a mechanism for proV-CATH cellular retention

The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA-proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA-green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA-proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA-proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH.     

The retrieval function of the KDEL receptor requires PKA phosphorylation of its C-terminus

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.     

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 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.     

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI)

We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.     

Improvement of Intracellular Traffic System by Overexpression of KDEL Receptor 1 in Antibody‐Producing CHO Cells

The localization of soluble endoplasmic reticulum (ER) chaperones in the cell organelle is mediated by the C‐terminal KDEL (lysine, aspartic acid, glutamic acid and leucine) motif. This motif is recognized by the KDEL receptor, a seven‐transmembrane protein that cycles between the ER and cis‐Golgi to capture missorted KDEL chaperones from post‐ER compartments in a pH‐dependent manner. The KDEL receptor's target chaperones have a substantial role in protein folding and assembly. In this study, the gene expression level of KDEL receptor 1 shows a moderate upregulation during either ER stress or growth of Chinese hamster ovary (CHO) cells in batch culture, while the ER chaperones show higher upregulation. This might indicate the possibility of saturation of the ER retention machinery or at least hindered retention during late stage batch culture in recombinant CHO cells. KDELR1 is overexpressed in a monoclonal antibody‐producing CHO cell line to improve the intracellular chaperone retention rate in the ER. An increase in the specific productivity of IgG1 by 13.2% during the exponential phase, and 23.8% in the deceleration phase of batch culture is observed. This is the first study to focus on the ER retention system as a cell engineering target for enhancing recombinant protein production.     

A C-terminal signal prevents secretion of luminal ER proteins

Proteins that permanently reside in the lumen of the endoplasmic reticulum (ER) must somehow be distinguished from newly synthesized secretory proteins, which pass through this compartment on their way out of the cell. Three luminal ER proteins whose sequence is known, grp78 ("BiP"), grp94, and protein disulphide isomerase, share the carboxy-terminal sequence Lys-Asp-Glu-Leu (KDEL). We show that deletion (or extension) of the carboxyl terminus of grp78 results in secretion of this protein when it is expressed in COS cells. Conversely, a derivative of chicken lysozyme containing the last six amino acids of grp78 fails to be secreted and instead accumulates in the ER. We propose that the KDEL sequence marks proteins that are to be retained in the ER and discuss possible retention mechanisms.     

Signals and mechanisms for protein retention in the endoplasmic reticulum

After their co-translational insertion into the ER lumen or the ER membrane, most proteins are transported via the Golgi apparatus downstream on the secretory pathway while a few protein species are retained in the ER. Polypeptide retention in the ER is either signal-independent or depends on specific retention signals encoded by the primary sequence of the polypeptide. A first category, i.e. the newly synthesized polypeptides that are unable to reach their final conformation, are retained in the ER where this quality control generally results in their degradation. A second category, namely the ER-resident proteins escape the bulk flow of secretion due to the presence of a specific N- or C-terminal signal that interacts with integral membrane or soluble receptors. ER retention of soluble proteins mediated by either KDEL, HDEL or related sequences and membrane receptors has been relatively well characterized in plants. Recent efforts has been relatively well characterized in plants. Recent efforts have aimed at a characterization of the retention signal(s) of type I membrane proteins in the plant ER.     

The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum retrieval in yeast

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.     

Beyond KDEL: the role of positions 5 and 6 in determining ER localization

The C-terminal amino acid sequence of a protein plays an important role in determining the endoplasmic reticulum (ER) localization of many soluble proteins that enter the secretory pathway. While it is known that the four amino acids found at the extreme C-terminus of the protein (e.g., KDEL) play a critical role in the interaction with the receptors that mediate retrograde transport back to the ER, other factors within the protein are less well known. Here we show that positions − 5 and − 6 play an important role in determining the ER localization of soluble proteins, with the amino acids at these positions playing an essential role in ER localization of the human protein disulfide isomerase family member, ERp18. Three other naturally occurring C-terminal motifs were also found that work efficiently in ER localization as six-amino-acid variants, but inefficiently as the four-amino-acid variant. Using bimolecular fluorescence complementation, we demonstrate that positions − 5 and − 6 from the C-terminus of the protein play an important role in the recognition of KDEL-like ER retrieval motifs, with the three different human KDEL receptors showing different specificities for changes at these positions for both inefficient and efficient ER localization four-amino-acid motifs.     

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.     

Abstract omitted from Cell Stress & Chaperones Volume 3 Supplement 1 September 1998: Enzymatic removal of the ER retention signal KDEL from heat shock proteins

We have identified a proteolytic activity in rat liver microsomes that specifically removes the C-terminus from a spectrum of ER chaperones. We refer to this activity as heat shock protein (hsp convertase (HSPC). All of the substrates for HSPC that we have identified are hsp, and contain the KDEL or KEEL at their C-termini, a signal sequence for ER retention. HSPC conversion of GRP94, ERp72 and calreticulin was rapid and no evidence of N-terminal alteration was detected. The conversion was unaffected by the presence of other membrane proteins. Two ER proteins that are very sensitive to non-specific proteases, cytochrome b5 and the NADH-cytochrome b5 reductase were also tested as substrates for the HSPC. SDS-PAGE and immunoblot analysis of the incubation mixture showed no alteration in the mobilities of the cytochrome b5 and its reductase. Lysomotropic agents leupeptin and pepstatin A were ineffective in inhibiting HSPC. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the teosome inhibitor lactacystin also failed to inhibit the HSPC activity. Specific enzymatic removal of the KDEL signal may represent a novel mechanism for the regulation of ER protein trafficking or the function of ER hsp. The discovery of HSPC may provide a biochemical explanation for observations that were previously attributed to the inefficiency of KDEL retention. Under special circumstances, hsp that are normally localized in the ER lumen are transported to the plasma membrane. The relocalization of ER hsps to the cell surface has been linked to malignant transformation and to apoptosis. ERp72 is expressed on the surface of human tumor cells, but is confined to the ER in normal cells. It is proposed that the physiological role of HSPC is to remove the ER retention signal from lumenal heat shock proteins thereby permitting the translocation of the modified chaperones into a variety of non-ER locales. Relocated, or modified chaperones may participate in cellular functions including protein degradation, antigen presentation, protein folding, cell adhesion, and the regulation of gene expression.     

Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein

Elongation of very long chain fatty acids 4 (ELOVL4) is a novel member of the ELO family of genes that are involved in fatty acid metabolism. ELOVL4 encodes a putative transmembrane protein of 314 amino acids that carries a possible endoplasmic reticulum (ER) retention/retrieval signal (KXKXX) at the C-terminus. Two distinct mutations, a 5-bp deletion and a complex mutation from the same region in exon 6 of this gene, have been reported so far and are associated with autosomal dominant atrophic macular degeneration (adMD/STGD3). Both of these deletions could result in C-terminal truncation and loss of the ER retention signal in the mutant protein. We expressed the wild-type and mutant proteins in COS-7 and CHO cells to study the intracellular distribution of ELOVL4 and to identify possible implications of the above mutations in its localization. Immunofluorescence analysis of these proteins along with organelle marker antibodies revealed predominant ER localization for wild-type ELOVL4. Targeted deletion of the dilysine motif at the C-terminus of the protein resulted in the loss of ER localization. Immunoelectron microscopy and immunofluorescence analysis revealed a similar ER localization pattern for the protein in human photoreceptors. These data indicate that ELOVL4 is an ER-resident protein, which supports its suggested function in fatty acid elongation. We also demonstrate that the localization of both mutant proteins was dramatically changed from an ER to a Golgi distribution. Our observations suggest that the consequences of defective protein trafficking could underlie the molecular mechanism associated with degeneration of the macula in the patients with adMD/STGD3.     

Depletion of Molecular Chaperones from the Endoplasmic Reticulum and Fragmentation of the Golgi Apparatus Associated with Pathogenesis in Pelizaeus-Merzbacher Disease

Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of hypomyelinating disorders, from mild spastic paraplegia type 2 to severe Pelizaeus-Merzbacher disease (PMD). Mutant PLP1 accumulates in the endoplasmic reticulum (ER) and induces ER stress. However, the link between the clinical severity of PMD and the cellular response induced by mutant PLP1 remains largely unknown. Accumulation of misfolded proteins in the ER generally leads to up-regulation of ER chaperones to alleviate ER stress. Here, we found that expression of the PLP1-A243V mutant, which causes severe disease, depletes some ER chaperones with a KDEL (Lys-Asp-Glu-Leu) motif, in HeLa cells, MO3.13 oligodendrocytic cells, and primary oligodendrocytes. The same PLP1 mutant also induces fragmentation of the Golgi apparatus (GA). These organelle changes are less prominent in cells with milder disease-associated PLP1 mutants. Similar changes are also observed in cells expressing another disease-causing gene that triggers ER stress, as well as in cells treated with brefeldin A, which induces ER stress and GA fragmentation by inhibiting GA to ER trafficking. We also found that mutant PLP1 disturbs localization of the KDEL receptor, which transports the chaperones with the KDEL motif from the GA to the ER. These data show that PLP1 mutants inhibit GA to ER trafficking, which reduces the supply of ER chaperones and induces GA fragmentation. We propose that depletion of ER chaperones and GA fragmentation induced by mutant misfolded proteins contribute to the pathogenesis of inherited ER stress-related diseases and affect the disease severity.