Figure 8.

The interaction between GLUP4/Rab5 and GLUP6/GEF as assessed by gel filtration analysis (A) and coimmunoprecipitation (B). A, The physical interaction between GST-GLUP4/Rab5 and GST-GLUP6/GEF in vitro was analyzed by gel filtration assay. The elution profiles, monitored by the A₂₈₀, are shown in GST-GLUP4/Rab5 (red), GST-GLUP6/GEF (green), and the mixture of both (blue). The peak positions of the marker proteins are indicated on the top, and the elution volumes and estimated molecular weights in parentheses are indicated above the peaks of each sample. Aliquots (12 μL) of each eluate fraction were subjected to 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. B, Coimmunoprecipitation of GLUP4/Rab5 and GLUP6/GEF. A total protein extract from developing seed was immunoprecipitated using the anti-GLUP4/Rab5 antibody (lanes 3 and 4) prior to analysis of the captured material by SDS-PAGE and western blotting using the anti-GLUP6/GEF antibody. Negative control reactions containing PBS (lane 1) or preimmune antibody (lane 2) in place of anti-GLUP4/Rab5 antibody are shown. Lanes 1 to 3 and 4 show the protein extract from the wild type and glup6 seeds, respectively. The high background seen in lanes 2 to 4 is due to the long exposures required to detect the Rab5-GEF immunoprecipitate. Rab5 is present in small amounts in developing endosperm.