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.     

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.     

Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.     

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.     

A C. elegans gene encodes a protein homologous to mammalian calreticulin.

The gene encoding a C. elegans homologue of the mammalian reticuloplasmin, calreticulin, was cloned and sequenced and the amino-acid sequence of its product deduced. The coding region of the gene comprises three exons separated by introns of 95 and 55 nucleotides, followed by either 158 or 279 bases of 3' non-coding sequence before putative polyadenylation signals. The precursor protein of 395 residues includes an N-terminal signal sequence of 13 residues. The C-terminus has the ER retention signal HDEL preceded by a polyacidic zone similar to known mammalian calreticulins. The sequence shows a 61% identity with mouse calreticulin, increasing to 82% in the proline-rich region of the molecule. Comparison of the C. elegans sequence with the calreticulin-related antigen RAL-1 of Oncocerca volvulus shows 73% identity, excluding the calreticulin C-terminal region. The sequence of this region differs markedly from RAL-1 where the parasite protein has a polybasic stretch and no ER retention signal. The C. elegans gene described here and designated crt-1 was mapped to a region towards the left-hand end of Chromosome V on the physical map of the genome. Southern blotting of genomic DNA indicates that in C. elegans the calreticulin homologue exists in only one form as the product of a single gene.     

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.     

Paralog of the formylglycine-generating enzyme--retention in the endoplasmic reticulum by canonical and noncanonical signals

Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the oxidation of a specific cysteine residue to formylglycine, which is the catalytic residue required for sulfate ester hydrolysis. This post-translational modification occurs in the endoplasmic reticulum (ER), and is an essential step in the biogenesis of this enzyme family. A paralog of FGE (pFGE) also localizes to the ER. It shares many properties with FGE, but lacks formylglycine-generating activity. There is evidence that FGE and pFGE act in concert, possibly by forming complexes with sulfatases and one another. Here we show that human pFGE, but not FGE, is retained in the ER through its C-terminal tetrapeptide PGEL, a noncanonical variant of the classic KDEL ER-retention signal. Surprisingly, PGEL, although having two nonconsensus residues (PG), confers efficient ER retention when fused to a secretory protein. Inducible coexpression of pFGE at different levels in FGE-expressing cells did not significantly influence the kinetics of FGE secretion, suggesting that pFGE is not a retention factor for FGE in vivo. PGEL is accessible at the surface of the pFGE structure. It is found in 21 mammalian species with available pFGE sequences. Other species carry either canonical signals (eight mammals and 26 nonmammals) or different noncanonical variants (six mammals and six nonmammals). Among the latter, SGEL was tested and found to also confer ER retention. Although evolutionarily conserved for mammalian pFGE, the PGEL signal is found only in one further human protein entering the ER. Its consequences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved.     

BmCREC Is an Endoplasmic Reticulum (ER) Resident Protein and Required for ER/Golgi Morphology

Silkworm posterior silkgland is a model for studying intracellular trafficking. Here, using this model, we identify several potential cargo proteins of BmKinesin-1 and focus on one candidate, BmCREC. BmCREC (also known as Bombyx mori DNA supercoiling factor, BmSCF) was previously proposed to supercoil DNA in the nucleus. However, we show here that BmCREC is localized in the ER lumen. Its C-terminal tetrapeptide HDEF is recognized by the KDEL receptor, and subsequently it is retrogradely transported by coat protein I (COPI) vesicles to the ER. Lacking the HDEF tetrapeptide of BmCREC or knocking down COPI subunits results in decreased ER retention and simultaneously increased secretion of BmCREC. Furthermore, we find that BmCREC knockdown markedly disrupts the morphology of the ER and Golgi apparatus and leads to a defect of posterior silkgland tube expansion. Together, our results clarify the ER retention mechanism of BmCREC and reveal that BmCREC is indispensable for maintaining ER/Golgi morphology.     

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots ( Arabidopsis , broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.     

Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Glucose-regulated GRP58 has shown clinical applications to endoplasmic reticulum (ER) stress and cancer. GRP58 is localized in the cytosol, endoplasmic reticulum (ER) and nucleus. Twenty-four amino acids at the N-terminal hydrophobic region are known to target GRP58 to ER for synthesis at the ER membrane and translocation into the ER lumen. In addition, GRP58 contains putative nuclear localization (494KPKKKKK500) and ER retention (502QEDL505) signals. However, the role of these signals in nuclear import and ER retention of GRP58 remains unknown. Present studies investigated the signals that control nuclear localization and ER retention of GRP58. Deletion/mutation of nuclear localization signal (NLS) abrogated nuclear import of GRP58. NLS attached to EGFP localized EGFP in the nucleus. However, deletion/mutation of putative ER retention signal alone did not alter ER retention of GRP58. Interestingly, a combined deletion/mutation of NLS and ER retention signals blocked the GRP58 retention in the ER. These results concluded that overlapping NLS and ER retention signal sequences regulate nuclear localization and ER retention of GRP58.     

Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.     

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.     

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.     

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.     

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.     

The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N- and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p-Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p-Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged efficiently into vesicles. We identified an isoleucine-leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was sufficient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine-tyrosine sequence required together with the isoleucine-leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p- Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a specific orientation for efficient packaging of the Erv41p-Erv46p complex.     

Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.     

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.     

The localization of the ER retrieval sequence for the calcium pump SERCA1

A number of studies using chimeric constructs made by fusing endoplasmic/sarcoplasmic reticulum calcium pump (SERCA) sequences with those of the plasma membrane located calcium pump (PMCA) have suggested that the retention/retrieval signal responsible for maintaining SERCA in the endoplasmic reticulum (ER) is located within the N-terminus of these pumps. Because of the difficulties in identifying the presence of constructs at the plasma membrane we have used a trans-Golgi network (TGN) marker to evaluate whether chimeric proteins are retained by the ER or have lost their retention/retrieval sequences and are able to enter the wider endomembrane system and reach the TGN. In this study, attempts to locate this retention/retrieval sequence demonstrate that the retention sequences are located not in the N-terminus, as previously suggested, but in the largely transmembranous C-terminal domain of SERCA. Further attempts to identify the precise retention/retrieval motif using SERCA1/PMCA3 chimeras were unsuccessful. This may be due to the fact that introducing SERCA1 sequences into the C-terminal PMCA3 sequence and vice versa disrupts the organization of the closely packed transmembrane helices leading to retention of such constructs by the quality control mechanisms of the ER. An alternative explanation is that SERCAs have targeting motifs that are non-linear, being made up of several segments of sequence to form a patch that interacts with the retrieval machinery.     

Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum

Recent studies have demonstrated the existence of glycosyl-phosphatidylinositol (GPI)-anchored proteins in higher plants. In this study we tested whether GPI-addition signals from diverse evolutionary sources would function to link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE') fused with a tobacco secretion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-terminus. The yeast and plant C-terminal signals were found to be capable of directing the addition of a GPI-anchor to the endoglucanase protein (EGE') as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was not linked to the EGE' protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addition signal sequences for yeast and plant cells.