RAB-D1 [N121I] inhibits secretory traffic between the ER and Golgi. (A)
Confocal images of tobacco leaf epidermal cells expressing the ER luminal
marker GFP-HDEL or the secreted GFP marker secGFP either alone or together
with wild-type or the N121I mutant forms of RAB-D1. (B) Quantification of the
effect of RAB-D1 and the indicated mutants on the intracellular accumulation
of secGFP (green bars). The fluorescence signal from the ER-lumenal marker
GFP-HDEL (blue bar) was set to 1 and that for secGFP alone to 0. Error bars
represent standard deviation of each sample. (C,D) Use of ratiometric
fluorescent polyproteins to localise secreted and Golgi markers in the
presence of RAB-D1 [N121I] and RAB-D2a [N121I]. (C) nlsR-2A-secG expresses
secGFP (green) and a nuclear-targeted RFP (red) in fixed stoichiometry as a
polyprotein linked by the self-cleaving 2A polypeptide
(Samalova et al., 2006).
Transfected cells are marked by red nuclei but do not accumulate GFP, which is
secreted to the cell wall (top). In the presence of RAB-D1 [N121I] (bottom) or
RAB-D2a [N121I] (centre), cells expressing similar quantities of the marker
have similarly bright RFP signals in their nuclei, but now secGFP accumulates
in a polygonal and planar ER network. (D) N-ST-R-2A-GH expresses the Golgi
marker N-ST-RFP (red) in fixed stochiometry with the ER-marker GFP-HDEL
(green), which indicates the relative expression level in each cell. In the
presence of RAB-D1 [N121I] and RAB-D2a [N121I], the Golgi marker accumulates
in the ER where it colocalises with GFP. In C and D, chlorophyll is blue.
Scale bars:100 μm in A; 10 μm in C and D.