Glycosylphosphatidylinositol-dependent protein trafficking in bloodstream stage Trypanosoma brucei

Abstract

We have previously demonstrated that glycosylphosphatidylinositol (GPI) anchors strongly influence protein trafficking in the procyclic insect stage of Trypanosoma brucei (M. A. McDowell, D. A. Ransom, and J. D. Bangs, Biochem. J. 335:681-689, 1998), where GPI-minus variant surface glycoprotein (VSG) reporters have greatly reduced rates of endoplasmic reticulum (ER) exit but are ultimately secreted. We now demonstrate that GPI-dependent trafficking also occurs in pathogenic bloodstream trypanosomes. However, unlike in procyclic trypanosomes, truncated VSGs lacking C-terminal GPI-addition signals are not secreted but are mistargeted to the lysosome and degraded. Failure to export these reporters is not due to a deficiency in secretion of these cells since the N-terminal ATPase domain of the endogenous ER protein BiP is efficiently secreted from transgenic cell lines. Velocity sedimentation experiments indicate that GPI-minus VSG dimerizes similarly to wild-type VSG, suggesting that degradation is not due to ER quality control mechanisms. However, GPI-minus VSGs are fully protected from degradation by the cysteine protease inhibitor FMK024, a potent inhibitor of the major lysosomal protease trypanopain. Immunofluorescence of cells incubated with FMK024 demonstrates that GPI-minus VSG colocalizes with p67, a lysosomal marker. These data suggest that in the absence of a GPI anchor, VSG is mistargeted to the lysosome and subsequently degraded. Our findings indicate that GPI-dependent transport is a general feature of secretory trafficking in both stages of the life cycle. A working model is proposed in which GPI valence regulates progression in the secretory pathway of bloodstream stage trypanosomes.

FIG. 1.

Diagram of secretory reporters. Diagrammatic representations of VSG 117, VSG 221, and BiP reporters are shown (scale approximate.) The hatched boxes denote N-terminal signal sequences. The filled circles indicate N-linked glycans. The black boxes signify the GPI attachment peptide, and the filled triangle shows the site of cleavage and GPI attachment to VSG. The native BiP structure is shown for comparison. The BiP ATPase and peptide binding domains are indicated.

FIG. 2.

Secretion of BiPN. (A) Bloodstream 221 cells expressing transgenic BiPN reporter were pulse-radiolabeled for 10 min with ³⁵S-labeled Met-Cys and then chased for 4 h. BiP polypeptides were immunoprecipitated from cell and medium fractions at the indicated times. Samples were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Each lane has 5 × 10⁶ cell equivalents. The positions of endogenous BiP, VSG, and the BiPN reporter have been indicated. (B) The experiment shown in panel A was performed in triplicate, and the amount of both the cell-associated and secreted forms of the two different BiPN reporters were quantified by phosphorimaging. Cell-associated (solid symbols) and medium-accumulated (open symbols; only the final datum point is shown) BiPN as a percentage of time zero (squares, means ± the standard error) are plotted as a function of chase time. The same analysis was also performed on a glycosylated version of BiPN (BiPN^CHO, triangles).

FIG. 3.

Fate of GPI-minus VSG. (A and B) Transgenic cell lines expressing GPI-minus VSG reporters 117Δgpi (A; endogenous VSG 221) or 221Δgpi (B; endogenous VSG 117) were pulse-chase radiolabeled as in Fig. 2. Immunoprecipitated VSG polypeptides were analyzed by SDS-PAGE and fluorography. All VSGΔgpi samples have 5 × 10⁶ cell equivalents per lane. Immunoprecipitations of endogenous VSGs (A; VSG 221, 5 × 10⁵ cell equivalents per lane; B, VSG 117, 2.5 × 10⁵ cell equivalents per lane) were analyzed as internal GPI-anchored controls. (C) Cell-associated (solid symbols) and medium-accumulated (open symbols) VSGΔgpi reporters (117Δgpi, squares; 221Δgpi, triangles) were quantified by phosphorimaging in three independent experiments. Cell-associated and secreted VSGΔgpis as a percentage of time zero (mean ± the standard error) are plotted as a function of the chase time.

FIG. 4.

Dimerization of GPI-minus VSG. Detergent extracts of radiolabeled transgenic bloodstream cells (5-min pulse, 5-min chase) were fractionated by velocity sedimentation. The sedimentation positions of transgenic 117Δgpi (A) and 221Δgpi (B) and endogenous, GPI-anchored VSG (data not shown) were determined by immunoprecipitation. Samples of each fraction were analyzed by SDS-PAGE, and the sedimentation positions of internal molecular mass standards (c = carbonic anhydrase, 31 kDa; b = bovine serum albumin, 68 kDa; γ = bovine gamma globulin, 170 kDa) were determined by Coomassie blue staining. The peak positions of internal markers and endogenous VSG are indicated. The scale refers to the molecular mass in kilodaltons.

FIG. 5.

Effect of FMK024 on VSGΔgpi degradation. Transgenic cell lines stably expressing 117Δgpi (A) or 221Δgpi (B) were pretreated for 1 h in the presence or absence of the proteasomal inhibitor lactacystin (1 μM) or the cysteine protease inhibitor FMK024 (117Δgpi, 2 μM; 221Δgpi, 20 μM). Cells were then radiolabeled (10-min pulse, 4-h chase) in the continued presence or absence of inhibitor. At the indicated chase times, aliquots (5 × 10⁶ cell equivalents) were removed and 117Δgpi (A) or 221Δgpi (B) was immunoprecipitated from cell (c) and medium (m) fractions. Samples were analyzed by SDS-PAGE and fluorography.

FIG. 6.

Localization of VSGΔgpi in bloodstream cells. Bloodstream cells expressing either 117Δgpi (A to H) or 221Δgpi (I to P) were incubated for 2 h in the absence (A, B, E, F, I, J, M, and N) or presence (C, D, G, H, K, L, O, and P) of FMK024 (117Δgpi, 2 μM; 221Δgpi, 20 μM). Fixed and permeabilized cells were stained as follows: panels E, G, M, and O, anti-BiP (red) and anti-VSGΔgpi (green); panels F, H, N, and P, anti-p67 (red) and anti-VSGΔgpi (green). All samples were counterstained with DAPI to reveal the nucleus (n) and the kinetoplast (k). Three channel merged images are presented in which colocalization is represented as yellow. The corresponding differential interference contrast-DAPI merged images are presented above each panel. The insets in panels F to H and panels N to P are the corresponding single channel images (p67, red; VSGΔgpi, green) in the region of the lysosome.