Volume 8 issue 1 January 2007
Rab10 is involved in basolateral transport in polarized MDCK cells
Sebastian Schuck, Mathias Gerl, Agnes Ang, Aki Manninen, Patrick Keller, Ira Mellman, Kai Simons

Figure S1. Accumulation of VSV-G in the Golgi is unaffected by Rab10Q68L. Coverslip-grown control and Rab10Q68L cells were infected with VSV-G adenovirus. VSV-G was accumulated in the ER at 39.5ºC for 6 h and chased out of the ER at 19.5ºC for 90 min. VSV-G co-localizes with giantin in control and Rab10Q68L cells (upper and lower panel, respectively), showing that it has left the ER and reached the Golgi. No change occurred when the incubation at 19.5ºC was prolonged to 120 min. Scale bars = 20 µm.

Figure S1 (.jpg)

Figure S2. Trypsin-based assay for biosynthetic sorting of VSV-G. Filter-grown control and Rab10Q68L cells were infected with VSV-G adenovirus. VSV-G was radioactively labeled for 9 h while accumulating in the ER at 39.5ºC and chased to the plasma membrane at 32ºC for 90 min. Cells were treated with trypsin from the apical or basolateral side at 4ºC for 30 min. Full-length VSV-G (VSV-G₀) and cleavage products (VSV-G_C) were precipitated with an antibody against the cytoplasmic YFP tag, separated by SDS-PAGE and quantified by phosphorimager analysis. The ratio of cleaved to full-length VSV-G was calculated for each sample, so that a measure for the fraction of VSV-G accessible by trypsin from the apical and basolateral side was obtained. Samples not treated with trypsin served as background controls.

Figure S2 (.jpg)

Figure S3. Activated Rab10 causes missorting of basolateral but not apical cargo. Coverslip-grown control and Rab10Q68L cells were infected with (A) VSV-G, (B) p75NTR or (C) apical VSV-G adenovirus. Cargo was accumulated for the minimum time necessary to allow microscopic analysis and chased to the plasma membrane at 32ºC (A, C) or 37ºC (B) for 90 min. Immunostaining for podocalyxin and, in (B), neurotrophin receptor. An apical and a basal optical section are shown for each condition. VSV-G is missorted to the apical membrane in the presence of Rab10Q68L, but the polarity of p75NTR and apical VSV-G is largely preserved. VSV-G is VSV-G ts045 (gray) with the YTDI sorting motif (red), fused to a spacer (black) and YFP (yellow). p75NTR is the human neurotrophin receptor (gray). Apical VSV-G is VSV-G ts045 (gray) fused to YFP (yellow) without a spacer, so that the basolateral sorting motif (red) is masked. Scale bars = 10 µm.

Figure S3 (.jpg)

Figure S4. Apical sorting of influenza virus hemagglutinin (HA) is essentially normal in the presence of activated Rab10. Filter-grown control and Rab10Q68L cells were infected with influenza virus and the polarity of HA surface transport was determined. The accuracy of HA targeting to the apical membrane is slightly reduced in cells expressing Rab10Q68L. This effect is minor compared to the severe missorting observed for VSV-G (see Figure 4B). Data are from an experiment performed in duplicate.

Figure S4 (.jpg)