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• W1993555998 abstract "Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family. Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family. The cell surface of the African trypanosome, Trypanosoma brucei, is dominated by glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; ISG, invariant surface glycoprotein; VSG, variant surface glycoprotein; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; HA, hemagglutinin; BiPN, N-terminal domain of BiP; ER, endoplasmic reticulum; TM, transmembrane; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole. 1The abbreviations used are: GPI, glycosylphosphatidylinositol; ISG, invariant surface glycoprotein; VSG, variant surface glycoprotein; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; HA, hemagglutinin; BiPN, N-terminal domain of BiP; ER, endoplasmic reticulum; TM, transmembrane; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole.-anchored proteins (1Grunfelder C.G. Engstler M. Weise F. Schwarz H. Stierhof Y.D. Boshart M. Overath P. Traffic. 2002; 3: 547-559Crossref PubMed Scopus (70) Google Scholar) and is composed of a single GPI-anchored polypeptide species, the variant surface glycoprotein (VSG) (2Ferguson M.A. Homans S.W. Dwek R.A. Rademacher T.W. Science. 1988; 239: 753-759Crossref PubMed Scopus (553) Google Scholar). VSG occupies most of the surface membrane area forming a densely packed monolayer (3Overath P. Stierhof Y. Wiese M. Trends Cell Biol. 1997; 1: 27-33Abstract Full Text PDF Scopus (121) Google Scholar, 4Mehlert A. Bond C.S. Ferguson M.A. Glycobiology. 2002; 12: 607-612Crossref PubMed Scopus (50) Google Scholar). Numerous proteins are present on the trypanosome cell surface in addition to VSG, albeit at lower densities, and include membrane transporters, proteases, and receptors (5El-Sayed N.M. Donelson J.E. J. Biol. Chem. 1997; 272: 26742-26748Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 6Burchmore R.J. Wallace L.J. Candlish D. Al-Salabi M.I. Beal P.R. Barrett M.P. Baldwin S.A. de Koning H.P. J. Biol. Chem. 2003; 278: 23502-23507Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 7Schell D. Evers R. Preis D. Ziegelbauer K. Kiefer H. Lottspeich F. Cornelissen A.W. Overath P.A. EMBO J. 1991; 10 (Correction (1993) EMBO J.12, 2990): 1061-1066Crossref PubMed Scopus (99) Google Scholar). Several families of cell surface proteins of unknown function, referred to as invariant surface glycoproteins (ISGs), are expressed exclusively in the bloodstream form (8Ziegelbauer K. Multhaup G. Overath P. J. Biol. Chem. 1992; 267: 10797-10803Abstract Full Text PDF PubMed Google Scholar, 9Ziegelbauer K. Overath P. J. Biol. Chem. 1992; 267: 10791-10796Abstract Full Text PDF PubMed Google Scholar, 10Jackson D.G. Windle H.J. Voorheis H.P. J. Biol. Chem. 1993; 268: 8085-8095Abstract Full Text PDF PubMed Google Scholar). ISG100 is a polytopic protein partly resident within the endosomal system (11Nolan D.P. Jackson D.G. Windle H.J. Pays A. Geuskens M. Michel A. Voorheis H.P. Pays E. J. Biol. Chem. 1997; 272: 29212-29221Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Pal A. Hall B.S. Nesbeth D.N. Field H.I. Field M.C. J. Biol. Chem. 2002; 277: 9529-9539Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), whereas the remainder of the characterized ISGs are type I transmembrane proteins with structural features similar to VSG (13Carrington M. Boothroyd J. Mol. Biochem. Parasitol. 1996; 81: 119-126Crossref PubMed Scopus (62) Google Scholar). The trypanosome surface is rapidly turned over by a highly active endocytic system, which serves to internalize nutrients, including transferrin and low-density lipoprotein via specialized receptors. Significantly, VSG is also rapidly recycled (1Grunfelder C.G. Engstler M. Weise F. Schwarz H. Stierhof Y.D. Boshart M. Overath P. Traffic. 2002; 3: 547-559Crossref PubMed Scopus (70) Google Scholar, 14Grunfelder C.G. Engstler M. Weise F. Schwarz H. Stierhof Y.D. Morgan G.W. Field M.C. Overath P. Mol. Biol. Cell. 2003; 14: 2029-2040Crossref PubMed Scopus (98) Google Scholar, 15O'Beirne C. Lowry C.M. Voorheis H.P. Mol. Biochem. Parasitol. 1998; 91: 165-169Crossref PubMed Scopus (65) Google Scholar), but the long half-life of VSG suggests that the protein is capable of multiple rounds of recycling (16Seyfang A. Mecke D. Duszenko M. J. Protozool. 1990; 37: 546-552Crossref PubMed Scopus (89) Google Scholar).The dominant mechanism of endocytosis in trypanosomes is mediated by clathrin, and this occurs exclusively from the flagellar pocket region (1Grunfelder C.G. Engstler M. Weise F. Schwarz H. Stierhof Y.D. Boshart M. Overath P. Traffic. 2002; 3: 547-559Crossref PubMed Scopus (70) Google Scholar, 17Allen C.L. Goulding D. Field M.C. EMBO J. 2003; 22: 4991-5002Crossref PubMed Scopus (178) Google Scholar). VSG is not concentrated during incorporation into clathrin-coated pits, probably because of the high density on the cell surface precluding further concentration. Significantly, T. brucei lacks the genes for both the AP-2 complex and an endocytic dynamin (14Grunfelder C.G. Engstler M. Weise F. Schwarz H. Stierhof Y.D. Morgan G.W. Field M.C. Overath P. Mol. Biol. Cell. 2003; 14: 2029-2040Crossref PubMed Scopus (98) Google Scholar, 18Morgan G.W. Hall B.S. Denny P.W. Carrington M. Field M.C. Trends Parasitol. 2002; 18: 491-496Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19Morgan G.W. Hall B.S. Denny P.W. Field M.C. Carrington M. Trends Parasitol. 2002; 18: 540-546Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 20Morgan G.W. Goulding D. Field M.C. J. Biol. Chem. 2004; 279: 10692-10701Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Selectivity is present as distinct cargo molecules are located in early endosomes as defined by Rab5A and Rab5B (12Pal A. Hall B.S. Nesbeth D.N. Field H.I. Field M.C. J. Biol. Chem. 2002; 277: 9529-9539Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The mechanism for sorting is not known, but we have suggested that segregation may be based on the mode of membrane attachment (12Pal A. Hall B.S. Nesbeth D.N. Field H.I. Field M.C. J. Biol. Chem. 2002; 277: 9529-9539Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A putative signal for delivery of the flagellar pocket protein CRAM to the cell surface has been reported (20Morgan G.W. Goulding D. Field M.C. J. Biol. Chem. 2004; 279: 10692-10701Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).Here we have used the ISG65 family to initiate investigation of transmembrane domain surface protein trafficking in bloodstream-form trypanosomes. Sequences related to defined tyrosine-containing endocytic signals, e.g. NPXXY and YKRF (21Yang H. Russell D.G. Zheng B. Eiki M. Lee M.G. Mol. Cell. Biol. 2000; 20: 5149-5163Crossref PubMed Scopus (11) Google Scholar), are not present. Hence, if ISG65 does contain internalization motifs, these are likely to be distinct from those in metazoans and yeast. The ISG65 family has a highly conserved C-terminal region, particularly in the transmembrane and cytoplasmic regions, and includes three conserved lysines. Interestingly, conserved lysine residues are also found in the cytoplasmic domain of ISG75 (22Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar) but not in the flagellar pocket-located transmembrane protein CRAM (20Morgan G.W. Goulding D. Field M.C. J. Biol. Chem. 2004; 279: 10692-10701Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To facilitate examination of the roles of the ISG65 C termini in protein targeting, we created a panel of fusion proteins containing the ISG65 C terminus attached to the N-terminal domain of BiP (23Ziegelbauer K. Rudenko G. Kieft R. Overath P. Mol. Biochem. Parasitol. 1995; 69: 53-63Crossref PubMed Scopus (14) Google Scholar). Further, creation of a set of deletion and substitution mutants allowed dissection of trafficking signals within the ISG65 cytoplasmic domain. In addition, we revisited the hypothesis that the mode of membrane attachment functions to direct proteins to specific early endosomal compartments.MATERIALS AND METHODSSource and Culture of Bloodstream Forms of T. brucei—Bloodstream forms of MITat 1.2, derived from Lister strain 427 and expressing VSG 221, were cultured in HMI-9 medium (24Bangs J.D. Brouch E.M. Ransom D.M. Roggy J.L. J. Biol. Chem. 1996; 271: 18387-18393Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), at 37 °C at 5% CO2 in medical flats (Corning) in a humid atmosphere. Continued expression of VSG 221 was checked periodically by immunofluorescence and always approached 100%. The cells were quantitated with a Z2 Coulter Counter (Coulter Electronics) and maintained at densities between 105 and 5 × 106 cells/ml. Mutant trypanosomes were maintained in HMI-9 selection medium with 2.5 μg/ml G418 (Sigma) or phleomycin (Invitrogen), as was appropriate, for expression of ectopic ISG constructs.Nucleic Acid and Recombinant DNA Methods—Standard molecular biology methods were carried out as described previously (25Hirumi H. Hirumi K. J. Parasitol. 1989; 75: 985-989Crossref PubMed Scopus (760) Google Scholar), unless otherwise indicated. Plasmids were grown in Escherichia coli XL1-Blue (Stratagene), unless otherwise described, following electroporation with a BTX600 ECM electroporator. PCR was performed using a 480 Thermal Cycler (PerkinElmer Life Sciences) with Herculase polymerase (Stratgene) and gel-embedded DNA purified using GeneClean (BIO101). The cloning vector pCR-Script was from Promega. The plasmids were prepared by using the Mini-prep spin kit (Qiagen). Oligonucleotide primers were obtained from Genosys and are detailed in Table I.Table ISequences of synthetic oligonucleotides used in this study. The following synthetic oligonucleotides were used for PCR. Sequences complementary to the target template are in upper case; additional sequences for cloning purposes are in lower case. Restriction sites are underlined, and mutagenic residues are in bold. All sequences are written 5′ to 3′.NameSequenceFwGpi117acggctagcAATGCTTGCAAAGATTCCTCTRevGpi117acggaattcctaAAAAAGCAAGGCCGCAAAFwGL117acggctagcTGCAAGAAGGAGAGCAACTGCFwTmacggctagcGATGCTGACTTTGACTTTGACGGGTTGRevTmacggaattcctaCATTACTACTTTTACGCTFwTmD1acggctagcGCAATGATTATATTGGCAGTCRevTmD2acggaattcttaCACCATTATGAAGAATGCRevTmD4acggaattcctaCTGGGAGTTATTCCTCCTRevTmD5acggaattcctaACCCTCCGCTTTTCCGGTFwTmK1acggctagcgATGCTGACTTTGACGGGTTGCTGGAGGCTGCCGAGGCTGCAGAGGTAACGCGTAGACATCAGCGTACGGCAATGRevTmK2acggaattcctaCATTACTACTTTTACGCTAGAAACCCCACCCTCCGCTTTTCCGGTGTCCACATCCTGGGAGTTATTCCGTCGACGCACCATRevTmK3acggaattcctaCATTACTACTTTTACGCTAGAAACCCCACCCTCCGCACGTCCGGTRevTmK4acggaattcctaCATTACTACGCGTACGCTRevTmK234acggaattcCTACATTACTACGCGTACGCTAGAAACCCCACCCTCCGCACGTCCGGTGTCCACATCCTGGGAGTTATTCCGTCGACGCACCATFwBiPNacggaattcAACCCGGGAATTATGRevBiPNacggaattcttagctagcCGCGTAATCTGGGACGTC Open table in a new tab Construction of BiPN-ISG/VSG Fusion Protein Genes—All chimeric BiPN synthetic genes are shown schematically in Fig. 1. The bloodstream-form expression vector pXS5BiPN, containing a Geneticin cassette and an HA9-tagged N-terminal (ATPase) BiP cassette (BiPN-HA9), was a gift generously given by J. Bangs, University of Wisconsin (23Ziegelbauer K. Rudenko G. Kieft R. Overath P. Mol. Biochem. Parasitol. 1995; 69: 53-63Crossref PubMed Scopus (14) Google Scholar). The C terminus of the BiPN-HA9 cassette was fused to either a GPI signal or transmembrane sequences using flanking NheI and EcoRI restriction sites. All these C-terminal signal sequences were generated by PCR from the genomic DNA of trypanosomes. The GPI signal sequence from VSG 117 (GPI(S)) was amplified using primers FwGpi117/RevGpi117. To ensure that processing of the GPI signal was not affected by being placed in an aberrant context, we also made a longer construct, GPI(L) with a 30-bp extension at the 5′-terminus, which was amplified using FwGL117/RevGpi117. The fragments were restricted by EcoRI and NheI and then ligated into pXS5BiPN to yield pXS5BiPNGPI(S) and pXS5BiPNGPI(L). Amplification of the C-terminal transmembrane (TM) sequence of ISG65 was performed with the primers FwTm/RevTm. The product was cloned into pXS5BiPN to generate pXS5BiPNTM. Sequencing confirmed that the construct corresponded to ISG65f and ISG65g isoforms (Fig. 1A). Deletions in the TM were made by reverse PCR of the whole pXS5BiPNTM. Three deletions in the cytoplasmic domain of TM were made with the forward primer FwBiPN and three reverse primers, RevTMD2, RevTMD4, and RevTMD5, to generate pXS5BiPNTMΔ49-74, pXS5BiPNTMΔ58-74, and pXS5BiPNTMΔ66-74, respectively. pXS5BiPNTMΔ1-23 was generated with primers FwTmD1/RevBiPN to remove the ISG ectodomain region. pXS5BiPNTMΔ1-23Δ49-74 was made with primers FwTMD1/RevBiPN on the template pXS5BiPNTMΔ1-23. For introducing Lys to Arg mutations, five fragments with lysine substitutions were generated from pXS5BiPNTM by using the following primers: FwTMK1/RevTM for Lys-1, FwTM/RevTMK2 for Lys-2, FwTM/RevTMK3 for Lys-3, FwTM/RevTMK4 for Lys-4, and FwTM/RevTMK234 for Lys-234. Fragments were restricted and ligated into prepared pXS5BiPN vector. All constructs were verified by standard sequencing methods (MWG Biotech) prior to introduction into trypanosomes and were further verified by Western blotting.Protein Electrophoresis and Western Blotting—Trypanosomes were harvested and washed twice in phosphate-buffered saline (Sigma). Pellets (107 cells) were lysed in 100 μl of boiling sample buffer and resolved by SDS-PAGE on 12% SDS-polyacrylamide minigels. The proteins were electrophoretically transferred onto nitrocellulose membranes (Amersham Biosciences) using a wet transfer tank (Hoefer Instruments). Equivalence of loading was verified by Ponceau Red (Sigma) staining of nitrocellulose membranes following transfer. Nonspecific binding was blocked with TBST (137 mm NaCl, 2.7 mm KCl, 25 mm Tris base, pH7.4, 0.2% Tween 20) supplemented with 5% freeze-dried milk. Antibodies were also diluted in TBST-milk. Rabbit polyclonal sera against anti-ISG65, anti-TbBiP (gift of J. Bangs, Madison, Wisconsin), and VSG 221 were diluted 1:10,000 in TBST-milk. A commercial monoclonal anti-HA9 (Santa Cruz Biotechnology) was used at 1:10,000. Incubations with commercial secondary anti-IgG rabbit or anti-IgG mouse horseradish peroxidase conjugates (Sigma) were performed at 10,000-fold dilution in TBST-milk. Bound antibodies were detected by chemiluminescence using the Amersham ECL detection system on Biomax MR-1 films (Kodak).Immunofluorescence Analysis—Indirect immunofluorescence microscopy was performed as described previously (26Ausubel F.M. Brent R. Kingston R.E. Moore D. Seidman J.G. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., 2003Google Scholar). Antibodies were used at the following dilutions: rabbit and mouse anti-HA9 epitope IgG (both from Santa Cruz Biotechnology) at 1:1000, rabbit anti-ISG65 at 1:1000, rabbit anti-TbRAB5A (anti-5A, 12) at 1:100, rabbit anti-TbRAB11A (anti-11, 27) at 1:1000, mouse anti-p67 (anti-p67, J. Bangs, Madison) at 1:1000, rabbit anti-BiP at 1:1000 and rabbit anti-VSG 221 at 1:1000. The anti-ISG65 antisera do not recognize the chimeric BiPNTM constructs, as only the extracellular region, not including the sequences expressed here, was used to raise the antisera. This was confirmed by Western analysis (data not shown). Secondary antibodies, anti-rabbit Cy3 (Sigma) and anti-mouse Oregon Green (Molecular Probes), were used according the manufacturer's instructions. The cells were examined on a Nikon Eclipse E600 epifluorescence microscope fitted with optically matched filter blocks and a Photometrics CoolSnap FX CCD camera. Digital images were captured at 24-bit gray scale using Metamorph V3 (Universal Imaging Corp.) on a WindowsXP computer (Microsoft Inc.), and the resulting images were false-colored, merged, and assembled in Adobe PhotoshopCS (Adobe Systems Inc.).Antibody Uptake—To stain for ISG65 and VSG 221 internalized into the cell, endocytosis of rabbit antiserum against either ISG65 or VSG 221 was allowed in living cells as follows. Bloodstream-form cells were harvested from 50-ml cultures at 1-2 × 106 cells/ml, pelleted at 800 × g for 10 min, washed twice in 10 ml of phosphate-buffered saline, and resuspended in 1 ml of ice-cold serum-free HMI-9. The cells were incubated with 20 μl of anti-ISG65 or 10 μl of anti-VSG 221 at 4 °C for 30 min, and the cells were washed three times with ice-cold TES at 14,000 rpm for 20 s in a refrigerated microcentrifuge (Eppendorf) and resuspended in HMI-9 and incubated for 0, 5, 10, and 20 min at 37 °C in 1.5-ml microtubes. The cells were again pelleted in the refrigerated microcentrifuge, and paraformaldehyde was immediately added to a final concentration of 3.5% for 30 min to fix the cells. The cells were then washed, spotted onto slides, permeabilized, blocked, and stained with Cy3-tagged anti-rabbit antibody as above.GPI-phospholipase C (GPIPLC) Assay—1 × 107 cells were resuspended in 200 μl of ice-cold water containing 0.1 mm TLCK and held on ice for 5 min. After centrifugation at 3,000 × g for 5 min, the supernatant was discarded. The cell ghosts were resuspended in 200 μl of 10 mm sodium phosphate buffer, pH 8.0, containing 0.1 mm TLCK. After incubation at 37 °C for 15 min, the sample was centrifuged at 16,000 × g for 15 min, and the supernatant (S) and pellets (P) analyzed by Western blotting (see Fig. 3). This protocol quantitatively solubilizes cell surface GPI-anchored VSG during the 37 °C incubation by activating endogenous GPI-phospholipase C, which is GPI-specific under these conditions (28Jeffries T.R. Morgan G.W. Field M.C. J. Cell Sci. 2001; 114: 2617-2626Crossref PubMed Google Scholar, 29Bangs J.D. Andrews N.W. Hart G.W. Englund P.T. J. Cell Biol. 1986; 103: 255-263Crossref PubMed Scopus (99) Google Scholar). Most cytoplasmic proteins are released in the initial 0 °C lysis step.Fig. 3BiPNGPI constructs contain a GPI anchor. Trypanosome lysates from untransfected parasites or cells expressing various BiPN constructs were subjected to a GPI-PLC release (see “Materials and Methods”), which converts GPI-anchored proteins to soluble forms (S). Uncleaved and transmembrane (TM) proteins are found in the pellet (P). VSG 221 and ISG65 were probed with the relevant antisera, and BiPN constructs were localized with anti-HA9. Migration positions of molecular mass markers are indicated at left in kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Protein Stability—Trypanosomes were cultured for up to 4 h in HMI-9 medium containing 50 μg/ml cycloheximide (Sigma) to prevent translation. The samples were taken at different time points and analyzed by Western blot. Data were quantitated by scanning of x-ray films following chemiluminescence exposure on a Heidelberg 1200 scanner, followed by analysis with ImageJ (rsbweb.nih.gov/ij/download.html).Bioinformatics—Sequences were aligned using ClustalW with default settings. Secondary and tertiary structure prediction and modeling was done using PSORT II (30Butikofer P. Ruepp S. Boschung M. Roditi I. Biochem. J. 1997; 326: 415-423Crossref PubMed Scopus (68) Google Scholar) and three-dimensional position-specific scoring matrix (31Nakai K. Horton P. Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1823) Google Scholar) software. Predicted structures were loaded into iMOL (www.pirx.com/iMol) for further analysis and presentation.RESULTSThe ISG65 Family Has a Conserved C-terminal Region— ClustalW alignment of the C-terminal regions of eight ISG65 family members indicated that, despite considerable variability within the ectodomains, the transmembrane and cytoplasmic regions are highly conserved (Fig. 1A). The predicted transmembrane regions are identical, and only a few conservative substitutions in the predicted cytoplasmic regions are found. Of additional interest was the presence of three fully conserved lysine residues in the cytoplasmic domain (red in Fig. 1A). Structural prediction using three-dimensional PSSM (31Nakai K. Horton P. Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1823) Google Scholar) suggested an open coiled structure for the cytoplasmic domain, with weak similarity to Tityus serrulatus toxin (Protein Data Bank, 1tsk) (32Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1120) Google Scholar). Interestingly, this predicted structure has lysine 62 fully exposed and therefore could present most of its sequence to cytosolic proteins for interaction (Fig. 1D).Design of ISG65 Reporter Constructs—ISG65 is most likely dimeric and encoded by a multigene family with a dimerization interface within the ectodomain. 2M. Carrington, unpublished data. This precludes the direct analysis of ISG65, as any mutant ISG65 isoform could form a heterodimer with endogenous ISG65. In addition, we have been unable to make ISG65 null mutants, and hence the experiments cannot be conducted in a null background. 2M. Carrington, unpublished data. Further, we wished to avoid any possibility of physiological interactions between the ISG65 ectodomain and other surface components. Therefore a set of chimeric reporter proteins were designed using the N-terminal domain of BiP (BiPN) (23Ziegelbauer K. Rudenko G. Kieft R. Overath P. Mol. Biochem. Parasitol. 1995; 69: 53-63Crossref PubMed Scopus (14) Google Scholar), incorporating an HA epitope tag (Fig. 1B). BiPN is rapidly secreted from trypanosomes, indicating that the protein folds efficiently (33Bangs J.D. Ransom D.M. McDowell M.A. Brouch E.M. EMBO J. 1997; 16: 4285-4294Crossref PubMed Scopus (56) Google Scholar). BiPN has the further advantage of not normally being present on the trypanosome cell surface.A full ISG65 transmembrane domain, including a short spacer region from the ectodomain to ensure preservation of correct membrane topology, was fused to BiPN (Fig. 1C, Bi-PNTM). A series of deletion constructs and substitution mutants, with one or more lysine residues changed to arginine, were also made (Fig. 1C). To compare the influence on intracellular transport of a GPI anchor versus a transmembrane domain, two GPI-anchored chimeric constructs containing the VSG 117 GPI signal sequence were made and designated Bi-PNGPI(S) and BiPNGPI(L). BiPNGPI(L) includes an additional eleven residues of the VSG C-terminal domain to allay concern that efficient processing at the ω site (underlined in Fig. 1B) could be prevented by close proximity to the BiPN domain (Fig. 1B).Expression of ISG65 Fusion Proteins in Trypanosomes—Following transfection and selection, lysates of transgenic parasites were analyzed by SDS-PAGE and Western blotting. Anti-HA monoclonals did not detect protein in wild-type trypanosomes but did react with lysates from cells transfected with both of the GPI-anchored BiPN chimeras (Fig. 2A). Interestingly, these proteins were expressed as more than one species; for BiPNGPI(L), the majority of immunoreactivity migrated at ∼62 kDa rather than at ∼50 kDa, as predicted from the polypeptide molecular mass. A low abundance species was detected at this position on the blot, suggesting that the decreased migration was because of post-translational modification. Further, the BiPNGPI(S) chimera also presented as two bands, but in this case, the major form was the lower molecular mass species. As BiPN lacks an N-glycosylation site, the increased molecular mass is probably due to processing of the GPI anchor core glycan (34Pinheiro C.B. Marangoni S. Toyama M.H. Polikarpov I. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 405-415Crossref PubMed Scopus (38) Google Scholar). Both isoforms in each case were efficiently solubilized by endogenous GPI-PLC (Fig. 3) and migrated with apparently lower molecular mass, consistent with removal of diacylglycerol. Hence BiPNGPI(L) and BiPNG-PI(S) likely contain glycolipid anchors.Fig. 2Expression of BiPN constructs in bloodstream-form trypanosomes. A, GPI-anchored BiPN constructs. Western blot analysis of trypanosomes. WT, parental; GPI L and S indicate parasites transfected with BiPNGPI(L) and BiPNGPI(S), respectively. The membrane was probed with anti-HA9 antibodies. Note the absence of reactivity in the parental. C designates the predicted migration position of the GPI-anchored polypeptide without further modification and M, the maturate forms. B, transmembrane BiPN constructs. Western blot analysis of trypanosomes expressing BiPNTM and derivatives. The upper panel blot was probed with anti-HA9 antibodies. BiPN constructs are designated as in Fig. 1. Lys-3 and Lys-4 refer to Lys to Arg substitutions. Blots were reprobed with anti-TbBiP (BiP) as a loading control. Migration positions of molecular mass markers are indicated at right in kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All of the lysine substitution mutants and most of the deletion mutants were also expressed in bloodstream-form trypanosomes (Figs. 2B and 5). These proteins migrated at the predicted molecular masses, and extensive post-translational modification is unlikely. The BiPNTM protein was also stably membrane-associated and fully resistant to solubilization by GPI-PLC (Fig. 3), consistent with the presence of a transmembrane anchor.Fig. 5Deletion of th" @default.
• W1993555998 created "2016-06-24" @default.
• W1993555998 creator A5059482371 @default.
• W1993555998 creator A5061440312 @default.
• W1993555998 creator A5078490247 @default.
• W1993555998 date "2004-12-01" @default.
• W1993555998 modified "2023-09-27" @default.
• W1993555998 title "Cytoplasmic Targeting Signals in Transmembrane Invariant Surface Glycoproteins of Trypanosomes" @default.
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