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Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei

David L. Alexander^*, Kevin J. Schwartz, Andrew E. Balber and James D. Bangs

The Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA
^* Present address: Department of Microbiology and Immunology, Stanford University Medical School, Fairchild Building, D305, Stanford, CA 94305, USA
Present address: StemCo Biomedical Inc., 2810 Meridian Parkway, Suite 148, Durham, NC 27713, USA

View larger version (24K): [in a new window]   Fig. 1. Vectors, peptide map and reporter constructs. (A) p67 processing events. The deduced topological structure (top) and the positions of the various glycoforms (bottom) are indicated. N-termini determined by direct peptide microsequencing are numbered by codon position within the deduced open reading frame. Black boxes, hydrophobic N-terminal signal sequence and C-terminal transmembrane domain; hatched box, glycosylated lumenal domain; open box, C-terminal cytoplasmic domain; lollipops, consensus N-glycosylation sites; dashed lines, uncertain cleavage sites. (B) Deduced amino acid sequence of the p67 transmembrane (black box) and C-terminal cytoplasmic domains. Acidic motifs are underlined and di-leucine motifs are in large font. The position of stop codons engineered to create the p67CD and p67TM reporter constructs are indicated. (C) Diagram of GFP fusion reporters. Black boxes, EP1 procyclin signal sequence and p67 transmembrane domain; gray box, EGFP orf; open box, p67 cytoplasmic domain. (D) The pXS5 stable expression vector. Segments from KpnI to SacI are labeled as described in Materials and Methods. Restriction sites are: K, KpnI; X, XhoI; C, ClaI; H, HindIII; R, EcoRI; S, SmaI; A, AscI; P, PacI, SI, SacI. Bar, 500 bp.

View larger version (48K): [in a new window]   Fig. 2. Processing of p67. (A-C) Pulse-chase analyses. Bloodstream (A,B) and procyclic (C) trypanosomes were pulse radiolabeled for 15 minutes and then chased in the absence (-) or presence (+) of the thiol protease inhibitor P27. At the indicated chase times p67 polypeptides were immunoprecipitated from cell extracts with mAb280 and analyzed by SDS-PAGE/fluorography. All lanes contain 107 cell equivalents. The mobilities of molecular mass markers in kDa and the positions of the various p67 glycoforms are indicated. (D) Procyclic (Pro) and bloodstream (BS) trypanosomes were metabolically radiolabeled for 9 and 4 hours, respectively, and p67 polypeptides were immunoprecipitated with mAb280. Immunoprecipitates were mock-treated (-) or PNG-treated (+) to remove N-glycans and analyzed by SDS-PAGE/fluorography. The positions of the native glycoforms (gp150 to gp32) and of the corresponding deglycosylated peptides (d150 to d32) are indicated. The origin of the smallest deglycosylated bloodstream polypeptide (?) is not known. All lanes contain 107 cell equivalents.

View larger version (65K): [in a new window]   Fig. 3. Localization of p67. Procyclic (A,E) and bloodstream (B and F, C and G, D and H) trypanosomes were fixed and permeabilized as described in Materials and Methods. Cells were stained with monoclonal mAb280 anti-p67 (E-H, red) and either rabbit anti-BiP (E,F, green), rabbit anti-trypanopain (G, green) or biotinyl-tomato lectin (H, green). Staining was visualized with appropriate secondary reagents and all samples were counterstained with DAPI (blue). Merged DIC and DAPI images (A-D) reveal the positioning of the large central nucleus (n) and compact posterior kinetoplast (k). Corresponding merged three channel fluorescent images are presented (E-H). Regions of colocalization appear yellow in merged images, and the matched single channel red (left) and green (right) images in the region of the lysosome are inset below for G and H. Arrowheads indicate regions of precise p67 co-localization with trypanopain and tomato lectin in G and H.

View larger version (53K): [in a new window]   Fig. 4. Endocytosis of receptor-mediated cargo. Bloodstream trypanosomes were incubated (1 hour, 37°C) with Alexa488-conjugated transferrin (A,B), biotinyl-tomato lectin (C,D), or biotinyl-tomato lectin and chitin hydrolysate (E,F) as described in Materials and Methods. Transferrin uptake was performed in the presence of 2 µM P27 to prevent degradation. Cells were then fixed, permeabilized and processed for fluorescence microscopy. Merged DIC/DAPI images (A,C,E) and corresponding merged three color fluorescent images (B,D,F) are presented (blue, DNA; red, p67; green, ligand). Regions of colocalization appear yellow in merged images and the matched single channel images of p67 (left) or ligand (right) staining in the region of the lysosome are inset at the bottom for each three channel image.

View larger version (44K): [in a new window]   Fig. 5. Biosynthesis and turnover of p67 reporter constructs. Procyclic (A,C) and bloodstream (B,D) cell lines stably expressing the truncated reporters, p67CD (A,B) and p67TM (C,D), were pulse radiolabeled (15 minutes) and then chased. At the designated times, samples (107 cell equivalents) were separated into cell and medium fractions and endogenous p67 polypeptides were removed by three rounds of immunoprecipitation with anti-CD antibody. p67 reporter polypeptides were then immunoprecipitated with mAb280 and analyzed as in Fig. 2. Panels A, B and D were generated by phosphorimaging and panel C by fluorography. Positions of the various p67 glycoforms are indicated.

View larger version (63K): [in a new window]   Fig. 6. Surface biotinylation of p67 reporter constructs. Untransformed (control) and transgenic (p67CD or p67WT) procyclic (A) and bloodstream (B) cell lines were surface biotinylated and cell lysates were subjected to specific immunoprecipitation with mAb280 anti-p67 (P), anti-hsp70 (H), and anti-transferrin receptor (T) antibodies. Immunoprecipitates were fractionated by SDS-PAGE, electrotransfered to membranes, blotted with HRP-strepavidin, and developed by chemiluminescence. All lanes contain 107 cell equivalents. Scale represents relative molecular mass in kDa.

View larger version (61K): [in a new window]   Fig. 7. Immunolocalization of p67 reporters. Transgenic procyclic (A-D) and bloodstream (E-H) cell lines expressing wildtype p67 (WT) or p67CD (CD) were fixed/permeabilized and immunostained with mAb280 to detect total p67 polypeptides (red). Cells were counterstained with DAPI (blue) to reveal nuclei and kinetoplasts. Matched sets of digitally merged DIC/DAPI and anti-p67/DAPI images are presented. All images were deconvolved and processed identically.

View larger version (43K): [in a new window]   Fig. 8. Expression of GFP reporters. (A) Stable cell lines expressing sGFPCD or sGFPWT were pulsed radiolabeled (15 minutes) in the absence (-) or presence (+) of trypanpain inhibitor and cell extracts were prepared at the indicated chase times. Reporter polypeptides were immunoprecipitated with anti-GFP and analyzed by phosphorimaging following SDS-PAGE. For quantitation the experiment was performed in triplicate and a representative image is presented. All lanes contain 5x106 cell equivalents.

View larger version (64K): [in a new window]   Fig. 9. Targeting of GFP reporters. Fixed/permeabilized procyclic cell lines expressing sGFPCD (CD) or sGFPWT (WT) were costained with specific antibodies for GFP (E-H, green) and BiP (E,G, red) or p67 (F,H, red). Cells were counterstained with DAPI (blue) to reveal nuclei and kinetoplasts. Matched DIC/DAPI (top) and three channel merged immunofluorescence (bottom) images are presented. Arrowheads indicate lysosomal regions of interest as discussed in the text. Single channel red (left) and green (right) images of the lysosomal region of each cell are inset in the bottom panels.