Figure 7.

Effect of AtRab1b(N121I) on Processing of N-Glycosylated GFP Derivatives.

(A) Extracts of leaf tissues expressing N-GFP-HDEL or N-ST-GFP were incubated either with (+) or without (−) endoH and analyzed by SDS-PAGE and immunoblotting with a GFP C-terminal antibody (N15; ABCAM). The N-glycans on GFP-HDEL, but not those on N-ST-GFP, are sensitive to removal by endoH, as illustrated by a shift of ∼2 kD in the apparent molecular mass of the major band.

(B) Cells expressing N-ST-GFP alone or N-ST-GFP plus AtRab1b(N121I) were analyzed as described for (A). Coexpression of N-ST-GFP with AtRab1b(N121I) resulted in the accumulation of an endoH-sensitive population of N-ST-GFP. This example shows that slightly less than half of the N-ST-GFP had accumulated in an endoH-sensitive form. Although in other experiments the endoH-sensitive population was sometimes slightly more abundant than the resistant form, it was never observed to be the predominant species.