Analysis of the signal for attachment of a glycophospholipid membrane anchor

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH-terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal.     

The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (omega) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.     

A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment

The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, does not become GPI anchored. We now show that this null sequence for GPI attachment can be converted to a strong GPI signal by mutating a pair of residues (valine-glutamate) in the LDLR sequence at a position corresponding to the normal cleavage/attachment site, to serine-glycine, as found in the DAF sequence. A single mutation (converting valine at the anchor addition site to serine, the normal acceptor for GPI addition in DAF) was insufficient to produce GPI anchoring, as was mutation of the valine-glutamate pair to serine-phenylalanine (a bulky residue). These results suggest that a pair of small residues (presumably flanking the cleavage point) is required for GPI attachment. By introducing the sequence serine-glycine (comprising a cleavage-attachment site for GPI addition) at different positions in the LDLR sequence of the fusion protein, HLD, we show that optimal GPI attachment requires a processing site positioned 10-12 residues NH2-terminal to the hydrophobic domain, the efficiency anchor attachment dropping off sharply as the cleavage site is moved beyond these limits. These data suggest that the GPI signal consists solely of a hydrophobic domain combined with a processing site composed of a pair of small residues, positioned 10-12 residues NH2-terminal to the hydrophobic domain. No other structural motifs appear necessary.     

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.     

Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.     

Isolation and characterization of the cell-associated region of group A streptococcal M6 protein.

DNA sequence analysis of the complete M6 protein gene revealed 19 hydrophobic amino acids at the C terminus which could act as a membrane anchor and an adjacent proline- and glycine-rich region likely to be located in the cell wall. To define this region within the cell wall and its role in attaching the molecule to the cell, we isolated the cell-associated fragment of the M protein. Assuming that the cell-associated region of the M protein would be embedded within the wall and thus protected from trypsin digestion, cells were digested with this enzyme, and the wall-associated M protein fragment was released by phage lysin digestion of the peptidoglycan. With antibody probes prepared to synthetic peptides of C-terminal sequences, a cell wall-associated M protein fragment (molecular weight, 16,000) was identified and purified. Amino acid sequence analysis placed the N terminus of the 16,000-molecular-weight fragment at residue 298 within the M sequence. Amino acid composition of this peptide was consistent with a C-terminal sequence lacking the membrane anchor. Antibody studies of nitrous acid-extracted whole bacteria suggested that, in addition to the peptidoglycan-associated region, a 65-residue helical segment of the C-terminal domain of the M protein is embedded within the carbohydrate moiety of the cell wall. Since no detectable amino sugars were associated with the wall-associated fragment, the C-terminal region of the M6 molecule is likely to be intercalated within the cross-linked peptidoglycan and not covalently linked to it. Because the C-terminal region of the M molecule is highly homologous to the C-terminal end of protein A from staphylococci and protein G from streptococci, it is likely that the mechanism of attachment of these proteins to the cell wall is conserved.     

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP^C into pathogenic PrP^Sc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.     

Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site.

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.     

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.     

An internally positioned signal can direct attachment of a glycophospholipid membrane anchor

All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.     

The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal-domain family found in Porphyromonas gingivalis

Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.     

Dependence of RGS9-1 membrane attachment on its C-terminal tail.

RGS9-1 is a GTPase-accelerating protein (GAP) required for rapid recovery of the light response in vertebrate rod and cone photoreceptors. Similar to its phototransduction partners transducin (G(t)) and cGMP phosphodiesterase, it is a peripheral protein of the disc membranes, but it binds membranes much more tightly. It lacks the lipid modifications found on G(t) and cGMP phosphodiesterase, and the mechanism for membrane attachment is unknown. We have used limited proteolysis to generate a fragment of RGS9-1 that is readily removed from membranes under moderate salt conditions. Immunoblots reveal that this soluble fragment lacks a 3-kDa fragment from the C-terminal domain, the only domain within RGS9-1 that differs in sequence from the brain-specific isoform RGS9-2. Recombinant fragments of RGS9-1 with or without the partner subunit G beta(5L) were constructed with or without the C-terminal domain. Those lacking the C-terminal domain bound to photoreceptor membranes much less tightly than those containing it. Removal by urea of G beta(5L) from endogenous or recombinant RGS9-1 bound to rod outer segment membranes left RGS9-1 tightly membrane-bound, and recombinant RGS9-1 was urea-soluble in the absence of membranes. Thus the C-terminal domain of RGS9-1 is critical for membrane binding, whereas G beta(5L) does not play an important role in membrane attachment.     

Expression, stability, and replacement of glucan-remodeling enzymes during developmental transitions in Saccharomyces cerevisiae

Sporulation is a developmental variation of the yeast life cycle whereby four spores are produced within a diploid cell, with proliferation resuming after germination. The GAS family of glycosylphosphatidylinositol-anchored glucan-remodeling enzymes exemplifies functional interplay between paralogous genes during the yeast life cycle. GAS1 and GAS5 are expressed in vegetative cells and repressed during sporulation while GAS2 and GAS4 exhibit a reciprocal pattern. GAS3 is weakly expressed in all the conditions and encodes an inactive protein. Although Gas1p functions in cell wall formation, we show that it persists during sporulation but is relocalized from the plasma membrane to the epiplasm in a process requiring End3p-mediated endocytosis and the Sps1 protein kinase of the p21-activated kinase family. Some Gas1p is also newly synthesized and localized to the spore membrane, but this fraction is dispensable for spore formation. By way of contrast, the Gas2-Gas4 proteins, which are essential for spore wall assembly, are rapidly degraded after spore formation. On germination, Gas1p is actively synthesized and concentrated in the growing part of the spore, which is essential for its elongation. Thus Gas1p is the primary glucan-remodeling enzyme required in vegetative growth and during reentry into the proliferative state. The dynamic interplay among Gas proteins is crucial to couple glucan remodeling with morphogenesis in developmental transitions.     

Sequence of the gene encoding flavocytochrome c from Shewanella putrefaciens: a tetraheme flavoenzyme that is a soluble fumarate reductase related to the membrane-bound enzymes from other bacteria.

Flavocytochrome c from the Gram-negative, food-spoiling bacterium Shewanella putrefaciens is a soluble, periplasmic fumarate reductase. We have isolated the gene encoding flavocytochrome c and determined the complete DNA sequence. The predicted amino acid sequence indicates that flavocytochrome c is synthesized with an N-terminal secretory signal sequence of 25 amino acid residues. The mature protein contains 571 amino acid residues and consists of an N-terminal cytochrome domain, of about 117 residues, with four heme attachment sites typical of c-type cytochromes and a C-terminal flavoprotein domain of about 454 residues that is clearly related to the flavoprotein subunits of fumarate reductases and succinate dehydrogenases from bacterial and other sources. A second reading frame that may be cotranscribed with the flavocytochrome c gene exhibits some similarity with the 13-kDa membrane anchor subunit of Escherichia coli fumarate reductase. The sequence of the flavoprotein domain demonstrates an even closer relationship with the product of the yeast OSM1 gene, mutations in which result in sensitivity to high osmolarity. These findings are discussed in relation to the function of flavocytochrome c.     

Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form

A cDNA was cloned coding for human placental 5'-nucleotidase. The 3547-bp cDNA contains an open reading frame that encodes a 574-residue polypeptide with calculated size of 63 375 Da. The NH2-terminal 26 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5'-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5'-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5'-nucleotidase.     

A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.     

The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment

Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl(-) channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.     

A glycophospholipid covalently attached to the C-terminus of the Thy-1 glycoprotein

The Thy-1 glycoprotein is a very abundant cell surface molecule of rat thymocytes and neuronal cells with the properties of a molecule that inserts into the lipid bilayer. The hydrophobicity is due to a glycophospholipid component covalently attached to the carboxy group of the C-terminal cysteine residue. The mature glycoprotein does not contain a stretch of hydrophobic amino acids that could traverse the membrane bilayer. These findings present a new mode of membrane attachment for a cell surface molecule that can mediate lymphokine release and cell division after cross-linking by antibodies.     

Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris

The aim of the study was the identification, cloning and disruption of the GAS1 homologue of Pichia pastoris. Gas1p is a glycoprotein anchored to the outer layer of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. Gas1p is a beta-1,3-glucanosyltransglycosylase (EC 2.4.1.-). This cross-linking enzyme highly affects the structure and permeability of the yeast cell wall. The gene coding for the GAS1 homologue of P. pastoris was cloned by PCR, and its functionality was proven in a Saccharomyces cerevisiae GAS1 null mutant. Based on the nucleotide sequence information of the P. pastoris GAS1 homologue, a disruption cassette was constructed for the knockout of the GAS1 in P. pastoris. The morphology of DeltaGAS1 P. pastoris was identical to that of S. cerevisiae GAS1 mutants. Finally, the impact of GAS1 disruption on secretion of three recombinant model proteins in P. pastoris, human trypsinogen, human serum albumin and Rhizopus oryzae lipase, was evaluated. While the disruption had no effect on the secretion of trypsinogen and albumin, the amount of lipase released from the cells was doubled.     

Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment

Glycophosphatidylinositol (GPI) membrane anchor attachment is directed by a cleavable signal at the COOH terminus of the protein. The complete lack of homology among different GPI-anchored proteins suggests that this signal is of a general nature. Previous analysis of the GPI signal of decay accelerating factor (DAF) suggests that the minimal requirements for GPI attachment are (a) a hydrophobic domain and (b) a cleavage/attachment site consisting of a pair of small residues positioned 10-12 residues NH2-terminal to a hydrophobic domain. As an ultimate test of these rules we constructed four synthetic GPI signals, meeting these requirements but assembled entirely from sequence elements not normally involved in GPI attachment. We show that these synthetic signals are able to direct human growth hormone (hGH), a secreted protein, to the plasma membrane via a GPI anchor. Our results indicate that different hydrophobic sequences, derived from either the prolactin or hGH NH2-terminal signal peptide, can be linked to different cleavage sites via different hydrophilic spacers to produce a functional GPI signal. These data confirm that the only requirements for GPI-anchoring are a pair of small residues positioned 10-12 residues NH2 terminal to a hydrophobic domain, no other structural motifs being necessary.