Figure 8.

Evidence That ERD2 Localization Is Restricted to Early Golgi Cisternae Even When Ligands Are Overexpressed.

(A) CLSM using higher resolution Airyscan detector showing strong colocalization of YFP-TM-ERD2 and RFP-TM-ERD2. Scatterplot and Spearman correlation coefficient were similar to data from conventional CLSM (Figure 7), confirming that both fusions can substitute for each other.

(B) CLSM using higher resolution Airyscan detector showing YFP-TM-ERD2 coexpressed with the Golgi-marker ST-RFP shows a clear segregation of structures labeled solely by ST-RFP (white arrowheads) as revealed by the distinct red population on the scatterplot and a significantly lower correlation coefficient.

(C) CLSM using higher resolution Airyscan detector of nonfunctional secYFP-ERD2 and functional RFP-TM-ERD2, revealing a very strong colocalization. Bars in (A) to (C) = 5 μm.

(D) CLSM of a typical transfected N. benthamiana protoplast with triple expression vector (Supplemental Methods 2) in dark field, showing the ERD2 localization in the presence of nonligand (Amy) versus ligand (Amy-HDEL) overexpression.

(E) Maximum intensity projection of a transfected protoplast in dark field (left) and bright field (right), showing no evidence of any green fluorescence in an ER network. Bars = 10 μm.

(F) Secretion index of the protoplast suspensions corresponding to (D) and (E), showing the expression of Amy-HDEL alone (con) or with YFP-TM-ERD2.

(G) Schematic drawing of early Golgi cisternae (G) connected by thin tubules (T), surrounded by an ER network (ER).