Supplemental Material

This article contains the following supporting material:

• Figure S1 - a) Schematic organization and luminal pH of the endocytic membrane trafficking pathways. The individual compartments have characteristic pHv (Mukherjee et al., 1997). b) Left panel: Endocytic rates of chimeras were measured by anti-CD4 Ab uptake in COS-7 cells as described in Methods. Right panel: Relative cell surface density of the chimeras was measured by Ab binding assay and normalized for cellular protein (means ± SEM, n=3)
• Figure S2 - Ubiquitin conjugation to CD4Tl-Ub. HEK293 stably expressing CD4Tl-Ub were transiently transfected with the indicated HA-Ub variants. CD4Tl-Ub was isolated by immunoprecipitation using rat anti-CD4 Ab in RIPA buffer. The precipitates were probed with anti-HA or polyclonal anti-CD4 Ab. Exogenous Ub expression was probed in the lysate by anti-HA Ab.
• Figure S3 - The effect of Ub overexpression on various cargo sorting. The postendocytic sorting of FITC-Tf, endogenous Lamp1 and CD4cc-UbR•G was monitoed by FRIA in cells overexpressing dsRed and wt Ub or UbR at a molar ratio of 1:10. Internalized Lamp1 and CD4 chimeras were labeled with anti-Lamp1 and anti-CD4 primary and FITC-conjugated secondary Ab as described in Fig. 4c. Only dsRed expressing cells were analyzed by FRIA. Data are representative of 2-3 independent experiments.
• Figure S4 - Lysosomal targeting of CD4 is facilitated by poly-Ub chain formation. Native CD4 was coexpressed with MARCH IV and Ub, UbR, UbR63K or UbR48K. Endocytic sorting was monitored by FRIA as in Fig. 4c with the modification that Abs were internalized for 1.5 h and chased for 90 min.
• Table S1