Volume 8 issue 8 August 2007
Multiple Motifs Regulate Trafficking of the LAMP-like Protein p67 in the Ancient Eukaryote Trypanosoma brucei
Ngii N. Tazeh and James D. Bangs

Supplemental Figure 1: Pulse-chase analysis of the ΔCD, D6 & D7 reporters.  Procyclic cell lines constitutively expressing the various reporters were pulse-chase radiolabeled as in Fig 2.  Samples were immunoprecipitated at 0 and 16 hours of chase, and analyzed by SDS-PAGE and phosphorimaging.  Cell lines expressing wild type (WT) and M3 reporters were used as controls.  The ΔCD reporter displayed faster mobility consistent with deletion of 20 residues.  Minor amounts of P1 fragment are seen with the D6 reporter (lane 6) and minor amounts of P2 fragment are seen with the D7 reporter (lane 8) consistent with lysosomal and cell surface trafficking, respectively.  The identities of F, P1, and P2 reporter species are explained in Fig. 2A.

Figure 1 (.jpg)

Supplemental Figure 2: Internal localization of the ΔCD, D6 & D7 reporters.  Procyclic cell lines were fixed, permeabilized and immunostained as in Fig 4.  Antibodies are anti-p67 (red) and anti-GFP (green).  Full cell 3-channel images (including DAPI stain, blue) are presented.  Typical images are presented, and the cell outlines are indicated by the dashed line.  Single channel red and green images of the lysosomal region are inset at the bottom of each 3-channel image.  All three reporters give precise ER colocalization with anti-BiP (data not shown).  The ΔCD reporter shows little evidence of lysosomal or surface localization.  The D6 reporter shows significant colocalization with the lyososomal marker p67 (arrowhead), and also apparent surface localization.  The D7 reporter shows no lysosomal localization but does appear on the cell surface.  Cell surface localizations are confirmed in Figs S3 and S4.  Scale bars indicate 5 microns.

Figure 2 (.jpg)

Supplemental Figure 3: Surface localization of the ΔCD, D6 & D7 reporters (immunofluorescence).  Procyclic cell lines were fixed without permeabilization, immunostained with anti-GFP (green), and imaged as in Fig 5.  Typical images are presented, and the cell outline for the ΔCD reporter is indicated.  Both the D6 and D7 cell lines gave cell surface signals, but the D6 reporter was phenotypically more heterogeneous (see Table I).  The ΔCD cell line was uniformly negative for surface staining.  Scale bars indicate 5 microns.

Figure 3 (.jpg)

Supplemental Figure 4: Surface localization of the ΔCD, D6 & D7 reporters (flow cytometry).  Flow cytometry of reporter cell lines was performed as described in Fig. 6 using the WT and M3 reporters as control negative and positive, respectively.  The mean fluorescent intensities (mean ± SEM, n = 2) are presented in bar graph format.  Signals for isotype controls and untransfected procyclic cells were the same as WT (data not shown).

Figure 4 (.jpg)