Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.     

Ethanolamine phosphate linked to the first mannose residue of glycosylphosphatidylinositol (GPI) lipids is a major feature of the GPI structure that is recognized by human GPI transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.     

Modified constructs of the tRNA TPsiC domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases

The TΨC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m⁵C₄₉, T₅₄, Ψ₅₅ and m¹A₅₈. U₅₄ is methylated to m⁵U (T) by m⁵U₅₄ methyltransferase (RUMT); A₅₈ is methylated to m¹A by m¹A₅₈ tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3′-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T₅₄ and Ψ₅₅), naturally occurring modifications at unnatural positions (m⁵C₆₀), altered sugar puckers (dU₅₄ and/or dU₅₅) or with disrupted U-turn interactions (m¹Ψ₅₅ or m¹m³Ψ₅₅). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T₅₄ increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U₅₄ was found to be an important determinant for the activities of both RAMT and RUMT.     

A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.     

Structural requirements for the recruitment of Gaa1 into a functional glycosylphosphatidylinositol transamidase complex

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized on membrane-bound ribosomes, translocated across the endoplasmic reticulum membrane, and GPI-anchored by GPI transamidase (GPIT). GPIT is a minimally heterotetrameric membrane protein complex composed of Gaa1, Gpi8, PIG-S and PIG-T. We describe structure-function analyses of Gaa1, the most hydrophobic of the GPIT subunits, with the aim of assigning a functional role to the different sequence domains of the protein. We generated epitope-tagged Gaa1 mutants and analyzed their membrane topology, subcellular distribution, complex-forming capability, and ability to restore GPIT activity in Gaa1-deficient cells. We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at approximately 17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain.     

Endoplasmic reticulum localization of Gaa1 and PIG-T, subunits of the glycosylphosphatidylinositol transamidase complex

After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1(282), a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1(282) had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1(282) appended to alpha2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1(282), and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins.     

Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system.

Assembly of glycosylphosphatidylinositol (GPtdIns)-anchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the C-terminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdIns-anchoring reaction, we used a small reporter protein that becomes GPtdIns-anchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with site-specific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdIns-anchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Nepsilon-(5-azido-2-nitrobenzoyl)-lysyl-tRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdIns-attachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass approximately 60 kDa, approximately 70 kDa, and approximately 120 kDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The approximately 70-kDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.     

Programmable cleavage of linear double-stranded DNA by combined action of Argonaute CbAgo from Clostridium butyricum and nuclease deficient RecBC helicase from E. coli

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecB^exo–C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecB^exo–C were thoroughly explored using 0.03–25 kb dsDNAs. When combined with RecB^exo–C, CbAgo could cleave targets located 11–12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecB^exo–C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecB^exo–C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.     

A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis

Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.     

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol‐anchored proteins through selective glycan enrichment

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI‐APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI‐APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. Here, we present methodology that permits identification of GPI‐APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC‐MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI‐APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI‐APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI‐APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI‐AP expression and dynamics.