Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.     

Retrieval of HDEL proteins is required for growth of yeast cells

The ERD2 gene of Saccharomyces cerevisiae encodes the receptor which retrieves HDEL-containing containing ER proteins from the Golgi apparatus. Viable erd2 mutants have been isolated that show no obvious HDEL-dependent retention of the luminal ER protein BiP, suggesting that retrieval of HDEL proteins is not essential for growth. However, cells that lack Erd2p completely have a defective Golgi apparatus and cannot grow. This observation led to the suggestion that the receptor had a second function, possibly related to its ability to recycle from Golgi to ER. In this paper we investigate the requirements for Erd2p to support growth. We show that mutations that block its recycling also prevent growth. In addition, we show that all mutant receptors that can support growth have a residual ability to retrieve BiP, which is detectable when they are overexpressed. Mere recycling of an inactive form of the receptor, mediated by a cytoplasmic KKXX sequence, is not sufficient for growth. Furthermore, saturation of the receptor by expression of an HDEL-tagged version of pro-alpha factor inhibits growth, even of strains that do not show obvious BiP retention. We conclude that growth requires the HDEL-dependent retrieval of one or more proteins, and that these proteins can be recognized even under conditions where BiP is secreted. Genetic screens have failed to identify any one protein whose loss could account for the Erd2p requirement. Therefore, a growth may require the retention of multiple HDEL proteins in the ER, or alternatively the removal of such proteins from the Golgi apparatus.     

ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus.

We have previously shown that the C-terminal sequence HDEL acts as a retention signal for luminal endoplasmic reticulum (ER) proteins in Saccharomyces cerevisiae, and that it is possible to isolate mutants that fail to retain an invertase fusion protein bearing this signal. Analysis of many such mutants defines two genes, ERD1 and ERD2. Cells lacking the ERD1 gene secrete the endogenous ER protein, BiP. Under normal growth conditions, the rate of secretion is equivalent to the rate at which wild-type cells secrete a modified form of BiP that lacks the HDEL signal altogether. Thus, erd1 cells show a profound disruption of the retention system. The mutant cells have no gross abnormality of their intracellular membrane system, but show defects in the Golgi-dependent modification of glycoproteins. We suggest that sorting of luminal ER proteins normally occurs in the Golgi, and that the function of ERD1 is required for the correct interaction of an HDEL receptor with its ligands. The sequence of ERD1 predicts a membrane protein with several transmembrane domains, a conclusion supported by analysis of ERD1-SUC2 fusion proteins.     

Changing the specificity of the sorting receptor for luminal endoplasmic reticulum proteins.

Luminal proteins of the endoplasmic reticulum (ER) share a common carboxy-terminal tetrapeptide which is necessary and sufficient for their retention in the ER. In animal cells this retention signal is usually KDEL, whereas the yeast Kluyveromyces lactis uses the closely related sequences HDEL and DDEL. The yeast ERD2 gene has been shown to determine the capacity and specificity of the retention system, implying that it encodes a sorting receptor. This receptor is thought to retrieve escaped ER proteins from the Golgi, where a human homologue of this protein has been located. This dual function of binding and retrieval requires a receptor with highly specific binding at a specific location in the cell (Golgi but not ER). Here, a region of the ERD2 protein responsible for the specificity of ligand recognition has been identified using three independent approaches. A single amino acid residue is shown to selectively affect HDEL retention: substitution of residue 51 of the K. lactis receptor is sufficient to abolish recognition of HDEL but not DDEL, generating a novel retention phenotype.     

Sorting of soluble ER proteins in yeast.

In animal cells, luminal endoplasmic reticulum (ER) proteins are prevented from being secreted by a sorting system that recognizes the C-terminal sequence KDEL. We show that yeast has a similar sorting system, but it recognizes HDEL, rather than KDEL: derivatives of the enzyme invertase that bear the HDEL signal fail to be secreted. An invertase fusion protein that is retained in the cells is partially modified by outer-chain mannosyl transferases, which reside in the Golgi element. This supports the view, based on studies in animal cells, that ER targeting is achieved by continuous retrieval of proteins from the Golgi. We have used an invertase fusion gene to screen for mutants that are defective in this sorting system. Over 60 mutants were obtained; eight of these are alleles of a single gene, erd1. The mutant strains grow normally at 30 degrees C, but instead of retaining the fusion protein in the cells, they secrete it.     

The Saccharomyces cerevisiae SEC20 gene encodes a membrane glycoprotein which is sorted by the HDEL retrieval system.

The SEC20 gene product (Sec20p) is required for endoplasmic reticulum (ER) to Golgi transport in the yeast secretory pathway. We have cloned the SEC20 gene by complementation of the temperature sensitive phenotype of a sec20-1 strain. The DNA sequence predicts a 44 kDa protein with a single membrane-spanning region; Sec20p has an apparent molecular weight of 50 kDa and behaves as an integral membrane protein with carbohydrate modifications that appear to be O-linked. A striking feature of this protein is its C-terminal sequence, which consists of the tetrapeptide HDEL. This signal is known to be required for the retrieval of soluble ER proteins from early Golgi compartments, but has not previously been observed on a membrane protein. The HDEL sequence of Sec20p is not essential for viability but helps to maintain intracellular levels of the protein. Depletion of Sec20p from cells results in the accumulation of an extensive network of ER and clusters of small vesicles. We suggest a possible role for the SEC20 product in the targeting of transport vesicles to the Golgi apparatus.     

ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway

Resident proteins of the ER lumen carry a specific tetrapeptide signal (KDEL or HDEL) that prevents their secretion. We have previously described the isolation of yeast mutants that fail to retain such resident proteins within the cell. Here we describe ERD2, a gene required for retention. It encodes a 26 kd integral membrane protein whose abundance determines the efficiency and capacity of the retention system. Reduced expression of ERD2 leads to secretion of proteins bearing the HDEL signal, whereas overexpression of ERD2 improves retention both in wild-type cells and in other mutants. These results are consistent with other evidence that ERD2 encodes the HDEL receptor (see accompanying paper). The gene is also required, perhaps indirectly, for normal protein transport through the Golgi, and hence for growth. We discuss possible roles for ERD2 in the secretory pathway.     

The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER

Proteins are co-translationally transferred into the endo-plasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (Δpro) forms of sporamin. The resulting SpoHDEL or ΔproHDEL, as well as Spo and Δpro, were expressed at high levels in transgenic tobacco cells ( Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or Δpro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or ΔproHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.     

The ERD2 gene determines the specificity of the luminal ER protein retention system

Luminal ER proteins carry a signal at their C terminus that prevents their secretion; in S. cerevisiae this signal is the tetrapeptide HDEL. Indirect evidence suggests that HDEL is recognized by a receptor that retrieves ER proteins from the secretory pathway and returns them to the ER, and a candidate for this receptor is the product of the ERD2 gene (see accompanying paper). We show here that presumptive ER proteins from the budding yeast K. lactis can terminate either with HDEL or, in the case of BiP, with DDEL. S. cerevisiae does not efficiently recognize DDEL as a retention signal, but exchange of its ERD2 gene for the corresponding gene from K. lactis allows equal recognition of DDEL and HDEL. Thus the specificity of the retention system is determined by the ERD2 gene. We conclude that ERD2 encodes the receptor that sorts luminal ER proteins.     

Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe.

We have cloned the gene for the resident luminal ER protein BiP from the fission yeast, Schizosaccharomyces pombe. The predicted protein product is equally divergent from the budding yeast and mammalian homologues. Disruption of the BiP gene in S. pombe is lethal and BiP mRNA levels are regulated by a variety of stresses including heat shock. Immunofluorescence of cells expressing an epitope-tagged BiP protein show it to be localized to the nuclear envelope, around the cell periphery and in a reticular structure through the cytoplasm. Unexpectedly, we find the BiP protein contains an N-linked glycosylation site which can be utilized. The C-terminal four amino acids of BiP are Ala-Asp-Glu-Leu, a new variant of the XDEL sequence found at the C-termini of luminal endoplasmic reticulum proteins. To determine whether this sequence acts as a sorting signal in S.pombe we expressed an acid phosphatase fusion protein extended at its C-terminus with the amino acids ADEL. Analysis of the sorting of this fusion protein indicates that the ADEL sequence is sufficient to cause the retention of proteins in the endoplasmic reticulum. The sequences DDEL, HDEL and KDEL can also direct ER-retention of acid phosphatase in S.pombe.     

Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.     

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.     

Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks

We have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jellyfish green fluorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves. All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks. Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum. This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin filaments. Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton. These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems. However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) significantly slowed down the recovery of Golgi fluorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.     

A novel Kex2 enzyme can process the proregion of the yeast alpha-factor leader in the endoplasmic reticulum instead of in the Golgi.

The prepro sequence of the yeast prepro-alpha-factor, usually referred to as the alpha-factor leader, has often been used for the efficient secretion of heterologous proteins from the yeast Saccharomyces cerevisiae. The alpha-factor leader consists of a 19-amino acid N-terminal pre or signal sequence followed by a 66-amino acid proregion. After removal of the signal sequence during membrane translocation, the proregion is cleaved from the precursor protein by the Kex2 endoprotease only in a late Golgi compartment. Here we report that a modified Kex2 enzyme, containing at the C-terminus the HDEL tetrapeptide, cleaves the proregion from the alpha-factor leader--human insulin like growth factor-1 fusion protein in the endoplasmic reticulum. The processing of pro-proteins earlier in the secretion pathway could be helpful in defining the cellular function of the proregions present naturally in various eucaryotic precursor proteins.     

Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum

Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.Mention of trademark, proprietary product, or vendor, does not constitute a guarantee or warrenty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations endoH endoglycosidase H - ER endoplasmic reticulum - Mr relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TBST Tris-buffered saline containing Tween 20 We thank Debra Donaldson for her contribution to the PHA gene constructions. This work has been supported by grants from the National Science Foundation (Cell Biology) and the Department of Energy (DE-FG03-86ER13497) to Maarten J. Chrispeels. The assistance of the staff of the Electron Microscope Laboratory, USDA, Beltsville is gratefully acknowledged.     

Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast

We demonstrate that a virally encoded yeast 'killer' toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted alpha/beta heterodimer that kills sensitive yeasts in a receptor-mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose beta-C-terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Deltaerd2) or mutants defective in Golgi-to-ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site-directed mutagenesis further indicated that the toxin's beta-HDEL motif ensures retrograde transport, although in a toxin-secreting yeast the beta-C-terminus is initially masked by an R residue (beta-HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high-level secretion of a biologically inactive protein incapable of re-entering the secretory pathway. Finally, we present evidence that ER-to-cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.     

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.     

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.     

Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein.

alpha 1,3 mannosyltransferase (Mnn1p) is a type II integral membrane protein that is localized to the yeast Golgi complex. We have examined the signals within Mnn1p that mediate Golgi localization by expression of fusion proteins comprised of Mnn1p and the secreted protein invertase. The N-terminal transmembrane domain (TMD) of Mnn1p is sufficient to localize invertase to the Golgi complex by a mechanism that is not saturable by approximately 15-20 fold overexpression. Furthermore, the TMD-mediated localization mechanism is clathrin dependent, as an invertase fusion protein bearing only the Mnn1p TMD is mislocalized to the plasma membrane of a clathrin heavy chain mutant. The Mnn1-invertase fusion proteins are not retained in the Golgi complex as efficiently as Mnn1p, suggesting that other signals may be present in the wild-type protein. Indeed, the Mnn1p lumenal domain (Mnn1-s) is also localized to the Golgi complex when expressed as a functional, soluble protein by exchanging its TMD for a cleavable signal sequence. In contrast to the Mnn1-invertase fusion proteins, overexpression of Mnn1-s saturates its retention mechanism, and results in the partial secretion of this protein. These data indicate that Mnn1p has separable Golgi localization signals within both its transmembrane and lumenal domains.     

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.