Fig. 4.

RAB-D1 and RAB-D2a act in ER-Golgi traffic via independent interactions. (A,B) Quantitation of secGFP accumulation (arbitrary units) in tobacco leaf epidermal cells expressing secGFP together with wild-type (wt) or N121I mutant forms (NI) of RAB-D1 (D1), RAB-D2a (D2a) or RAB8-E1d (E1d) alone or in combination as indicated. Error bars represent s.e.m. Values that are not significantly different (P≥0.05) are marked by an asterisk. Values with one asterisk are significantly different from values with two asterisks (P≤0.05). (A) Restoration of traffic inhibited by RAB-D2a [N121I]. (B) Restoration of traffic inhibited by RAB-D1 [N121I]. (C) Immunoblot analysis of tobacco leaves transiently expressing wild-type or mutant forms of RAB-D1 or RAB-D2a (as in A and B) using an anti-RAB-D2 antibody (see Materials and Methods). In uninfected plants (f) this antibody recognises two bands, most probably tobacco RAB-D proteins, the lower of which (black arrow) co-migrates with transiently expressed Arabidopsis RAB-D2a (lanes a, c and e). Note that the RAB-D2a [N121] mutant is not significantly overexpressed under these conditions and that it accumulates less efficiently than the wild type, consistent with previous observations that small GTPases are unstable in the nucleotide-free state when not bound to the exchange factor (Okkonen and Stenmark, 1997; Cool et al., 1999).