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First published online 15 November 2005
doi: 10.1242/jcs.02667

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GPI valence and the fate of secretory membrane proteins in African trypanosomes

¹ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA
² Department of Biomolecular Chemistry, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA

View larger version (19K): [in a new window]   Fig. 1. Diagram of reporter constructs. (A) Diagram of the full length EPMH reporter (above). Native EP1 procyclin domains (from N to C terminus) are: signal sequence, black box; mature N-terminal domain, white box, EP repeat domain, stripped box; GPI attachment peptide, gray box. The single EP1 N-glycosylation site (lollipop) and the C-terminus of the EPMHgpi reporter (arrowhead) are indicated. The position (codon 50) and sequence of the inserted cassettes are indicated (below). The insert sequence is in upper case, the methionine and HA cassettes are highlighted in black, and flanking native sequences are in lower case. (B) Domains and N-glycosylation (lollipops) patterns of the p67HP and BiPNHP GPI1 reporters: native signal sequences, black boxes; ectoplasmic domains, white boxes. The C-terminal HA and EP procyclin GPI-peptide sequences are indicated by dots. BiPNHP and p67HP contain codons 1-415 and 1-616 of the native sequences, respectively. (C) C-terminal amino acid sequence of the p67HP and BiPNHP GPI1 reporters including the HA epitope (underlined) and the EP procyclin GPI attachment peptide (black box). * indicates GPI attachment site.

View larger version (59K): [in a new window]   Fig. 2. Expression of EPMH reporters in bloodstream trypanosomes. (A) Control untransformed bloodstream cells (BS221) and cell lines expressing EPMH or EPMHgpi were preincubated (1 hour) with (+) or without (-) tunicamycin, and then metabolically radiolabeled with [35S]Met/Cys (1 hour) in the continued absence or presence of inhibitor. Reporters and endogenous VSG were immunoprecipitated from cell and media fractions with anti-EP9 (top, 2x107 cell equivalents/lane) and anti-VSG221 (bottom, 2x105 cell equivalents/lane) antibodies. Mobilities of the precursor (P), and mature (M) forms of EPMH, of EPHMgpi (), and of VSG (V), are indicated on the left; mobilities of molecular mass markers on the right. (B) EPMH cells were radiolabeled with [35S]Met/Cys for 2 hours and then immunoprecipitated with anti-EP9 (lane 1). Identical anti-EP9 immunoprecipitates were solubilized and reprecipitated with either anti-EP9 (lane 2) or biotinyl-tomato lectin (lane 3). All lanes contain 2x107 cell equivalents/lane. (C) EPMH cells were radiolabeled with [3H]Myristate for 4 hours and labeled polypeptides were immunoprecipitated from cell and media fractions with anti-EP9 (5x107 cell equivalents/lane) or anti-VSG221 (1x106 cell equivalents/lane). All samples were analyzed by SDS-PAGE and phosphorimaging (panels A and B) or fluorography (panel C).

View larger version (95K): [in a new window]   Fig. 3. Turnover of EPMH reporters in bloodstream trypanosomes Transgenic bloodstream cell lines expressing either EPMH (panel A) or EPMHgpi (panel B) were pulse radiolabeled with [35S]Met/Cys for 15 minutes and then chased with complete media. At the indicated times polypeptides were immunoprecipitated from cell and media fractions with either anti-EP9 (2x107 cell equivalents) or anti-VSG (2x105 cell equivalents). Samples were analyzed by SDS-PAGE and phosphorimaging. (A) `M' and `P' indicate the mobility of mature and precursor EPMH polypeptides. (B) As indicated, the EPHMgpi cells were either untreated or treated with FMK024 (20 µM) for 15 minutes prior to radiolabeling, and subsequently for the duration of the pulse/chase periods. Vertical white stripes indicate irrelevant regions of phosphoimages that were digitally removed to simplify presentation. The mobilities of molecular mass markers are indicated in kDa.

View larger version (62K): [in a new window]   Fig. 4. Immunolocalization of EPMH. (A-P) Methanol/acetone fixed bloodstream cells expressing the GPI1 EPMH reporter were stained with the indicated primary antibodies and visualized by epifluorescence microscopy. (A-D) Anti-EP9 and anti-HA. (E-H) Anti-EP9 and anti-BiP. (I-L) Anti-HA and anti-p67. (M-P) Live trypanosomes were exposed to biotinyl-tomato lectin (TL) at 5°C to allow flagellar pocket binding. After washing, cells were fixed and stained with anti-HA and anti-paraflagellar rod (PFR) antibodies. Paraflagellar rod and tomato-lectin staining were visualized simultaneously in the red channel with appropriate secondary reagents. Three consecutive Z-slices were merged to generate the red channel image. (Q-T) Cells were formaldehyde fixed without permeabilization and stained with anti-HA and anti-EP9. All panels: left column, merged DIC/DAPI images; right column, merged three channel fluorescent images (arrowheads indicate flagellar pocket localization). DAPI staining (blue) reveals nucleus (n) and kinetoplast (k) localization (indicated in panel A only).

View larger version (100K): [in a new window]   Fig. 5. Surface localization of EPMH by immuno-electron microscopy. Fixed cells were stained with monoclonal anti-EP antibody and colloidal gold-conjugated secondary antibodies prior to silver enhancement and preparation for electron microscopy. Untransformed BS221 cells (A) were used as a negative control for cell lines expressing EPMH (B-D), Note that this fixation technique does not allow detection of reporters in the lumen of the flagellar pocket (FP).

View larger version (98K): [in a new window]   Fig. 6. Immunolocalization of EPMHgpi. Bloodstream cells expressing EPMHgpi were mock-treated (panels A-L; -) or cultured with FMK024 for 1 hour (panels M-T; +). Cells were fixed/permeabilized, stained with the indicated antibodies, and visualized by epifluorescence microscopy. (A-D,M-P) Stained with anti-EP9 and anti-BiP antibodies. (E-L,Q-T) Stained with anti-EP9 and anti-p67 antibodies. All panels: left column, merged DIC/DAPI images; right column, merged three channel fluorescent images (arrowheads indicate lysosomal localization). DAPI staining (blue) reveals nucleus (n) and kinetoplast (k) localization (indicated in A only).

View larger version (50K): [in a new window]   Fig. 7. Fate of other GPI1 reporters. Bloodstream trypanosome cell lines expressing BiPNHP (A,B) or p67HP (C,D) in the absence (-) or presence (+) of FMK024, were pulse (15 minutes)-chase radiolabeled. Cell and media fractions were immunoprecipitated with specific anti-BiP (A,B) or anti-HA (C,D). All samples were analyzed by SDS-PAGE and phosphorimaging. Mobilities of precursor (P) and mature (M) forms of the BiPNHP and p67HP reporters, and of endogenous full-length BiP are indicated on the left of A-D.

View larger version (60K): [in a new window]   Fig. 8. Upregulation of TfR expression. Bloodstream 221 cells were pulse-chase radiolabeled as in Fig. 7 in the absence (A) or presence (B) of FMK024, and cell and media fractions were prepared at the indicated times. TfR polypeptides were immunoprecipitated with anti-TfR and analyzed by SDS-PAGE and phosphorimaging. (C) Bloodstream 221 cells were cultured 48 hours in HMI9 media containing 10% fetal bovine serum (FBS), 10% canine serum (CS), or 10% canine serum plus 200 µg/ml bovine transferrin (CS+). Cells were then pulse labeled (15 minutes) and chased under the same conditions. Cell (c) and media (m) fractions were prepared at 0 and 4 hours and analyzed as above. Fractional recovery of released ESAG7 polypeptide for each set is presented below (boxed). The relative amounts of initial ESAG7 are 1.0:14.0:6.1 for FBS (lane 1):CS (lane 5):Cs+ (lane 9). Distortions of lanes 5-12 are reproducible and probably result from non-specific precipitation of components of dog serum in lanes 6, 8, 10 and 12. (A-C) Mobilities of mature and immature forms of ESAG6 (E6m and E6i) and ESAG7 (E7) are indicated on the left. Mobilities of molecular mass standards are indicated on the right. All lanes contain 107 cell equivalents.

View larger version (173K): [in a new window]   Fig. 9. Surface localization of TfR. Bloodstream 221 cells were cultured 48 hours in HMI9 media containing 10% fetal bovine serum (BS, panels A,D,G) or 10% canine serum (CS, panels B,C,E,F,H,I). (A-C) Fixed cells were stained with anti-TfR antibody and colloidal gold-conjugated secondary antibodies prior to silver enhancement and preparation for electron microscopy. Note that this fixation technique does not allow detection of TfR in the lumen of the flagellar pocket (FP). (D-I) Live cells were incubated with Tf:gold (5 nm) conjugate at 8°C and prepared for electron microscopy as described in Materials and Methods. 1 mg/ml bovine holotransferrin was included in F,I as a competition control for specific binding. Arrowheads indicate endosomal elements containing Tf:gold. Size scales are indicated in µm. Selected regions of panels D, E and F are magnified 2x in panels D', E' and F', respectively.