A Diacidic Motif in the N-Terminal Cytosolic Domain of GONST1 Influences Its ER Export.
(A) Scheme of the topology of GONST1, with the sequence of the 30 N-terminal amino acids. Note the EHD and DLE motifs (underlined).
(B) GONST1-GFP localizes to punctate structures in tobacco leaf epidermal cells.
(C) to (E) The larger GONST1-labeled structures colocalize with ERD2-YFP (arrowheads), whereas the smaller structures are not labeled by ERD2-YFP (arrows).
(F) Mutation of DLE to GLA in GONST1 results in impaired ER export. Transport of GONST1^DXE-GFP out of the ER is reduced compared with that of the wild-type protein. Note the increase in ER staining, although some punctate structures remain visible (arrowheads).
(G) to (I) Coexpression of GONST1^DXE-GFP (G) with ERD2-YFP (H) confirms that the larger punctate structures labeled by GONST1^DXE-GFP are Golgi bodies (arrowheads). Note the colocalization of the two fluorochromes on dots in the merged image (I).
Bars = 5 μm.

Deletion of the N-Terminal Cytosolic Domain of GONST1 Does Not Abolish Its ER Export.
(A) Expression of GONST1^Δcyt-GFP in tobacco leaf epidermal cells shows increased ER staining compared with that in the wild-type protein (see Figures 1B to 1E), although some punctate structures remain visible (arrowhead), similar to the result observed with GONST1^DXE-GFP (see Figures 1F to 1I).
(B) to (D) The larger punctate structures labeled by GONST1^Δcyt-GFP (B) are Golgi bodies, as confirmed by coexpression of ERD2-YFP (C). Note the colocalization of the two fluorochromes on large dots (arrowhead) but not on small dots (arrow) in the merged image (D).
Bars = 5 μm.

Quantification of ER Fluorescence for GONST1-GFP and Its Mutants.
The fluorescence of the ER relative to that of the Golgi was quantified for each of the GONST1-GFP derivatives. The relative ER fluorescence (REF) is given as a percentage of the ratio between fluorescence intensity values (arbitrary units) measured in the ER and the sum of intensity values for the ER and Golgi (see Methods). Error bars represent sd for 21 measurements.

The Cytosolic Domain of CASP Contains Potential Diacidic ER Export Signals.
(A) Scheme of an N-terminal fusion of GFP to the Golgi matrix protein CASP. The GFP moiety and coiled-coil domain are shown as gray ellipses, and the transmembrane domain (TM) is shown as a black rectangle. The N terminus of the protein is cytosolic, whereas the C terminus is found in the lumen of the secretory pathway, giving the protein a type II topology (Renna et al., 2005). Part of the amino acid sequence of the cytosolic domain of CASP is shown (Renna et al., 2005). This domain contains two DXE motifs (underlined), which were mutated independently to GLG (DXE1) and GVG (DXE2).
(B) to (D) GFP-CASP localizes to the Golgi apparatus ([B] and [C]) (Renna et al., 2005), as demonstrated by its colocalization with ERD2-YFP (D).
(E) Merged image of (C) and (D).
(F) to (I) Mutation of DLE to GLG (DXE1) results in the redistribution of a large proportion of the fluorescence to the ER ([F] and [G]), although some punctate structures are visible ([F], arrowheads). Coexpression of ERD2-YFP (H) confirms that these structures are Golgi bodies through colocalization (I).
(J) to (M) Mutation of DVE to GVG (DXE2) has no obvious effect on ER–Golgi trafficking of the protein ([J] and [K]). Golgi bodies alone remain visible, confirmed by the colocalization of ERD2-YFP (L) with GFP-CASP^DXE2 (M).
Bars = 5 μm.

Quantification of ER Fluorescence for GFP-CASP and Its Mutants.
The fluorescence of the ER relative to that of the Golgi was quantified for each of the GFP-CASP derivatives. The relative ER fluorescence (REF) is given as a percentage as described for Figure 3. Error bars represent sd for 21 measurements.

A Synthetic Cargo Protein for Studying the Properties of the CASP Cytosolic Tail.
(A) Comparison of the topologies and domains of GFP-CASP, spNGFP-TM17, and spNGFP-TMcCCASP. The glycosylation site on GFP is represented by a short branch, and the transmembrane domain is shown as a black rectangle for each protein. The X represents the cleavage site for the signal peptide. The reverse orientation of the CASP tail in TMcCCASP is denoted by an arrow indicating the N-terminal to C-terminal direction.
(B) Proteinase K treatment of microsomes containing either spNGFP-HDEL or TMcCCASP. Note that the major band detected for TMcCCASP (lane 7) exhibits a shift to a lower molecular mass on treatment with proteinase K (lane 8, arrowhead), indicating that the GFP moiety of the protein is found in the lumen of the secretory pathway. spNGFP-HDEL does not show the same shift in molecular mass on treatment with the proteinase because it is contained within the lumen of the ER (cf. lanes 3 and 4). Both proteins become undetectable when the membranes are disrupted with Triton X-100 (lanes 6 and 10). UT, untransformed sample.

Export Signals Can Override Transmembrane Domain Length in Determining Destinations within the Secretory Pathway.
(A) spNGFP-TM17 is found exclusively in the ER, as a result of its short transmembrane domain.
(B) TMcCCASP localizes mainly to punctate structures, although some ER localization is also visible (arrowheads).
(C) to (E) Coexpression of TMcCCASP (C) with ERD2-YFP (D) demonstrates that the punctate structures labeled by TMcCCASP are Golgi bodies. Note the colocalization shown in the merged image (E).
(F) TMcCCASP^DXE1 is retained in the ER, in contrast with TMcCCASP.
(G) Export of TMcCCASP^DXE2 from the ER is apparently unaffected by the mutation.
Bars = 5 μm.

Quantification of ER Fluorescence for TMcCCASP and Its Mutants.
The fluorescence of the ER relative to that of the Golgi was quantified for each of the TMcCCASP derivatives. The relative ER fluorescence (REF) is given as a percentage as described for Figure 3. Error bars represent sd for 21 measurements.

TMcCCASP^DXE1 Does Not Travel to the Same Endomembrane Compartment as TMcCCASP.
EndoH treatment of protoplasts expressing spNGFP-HDEL, TMcCCASP, or TMcCCASP^DXE1. Although the glycan on TMcCCASP is resistant to digestion by EndoH (lane 8), that on TMcCCASP^DXE1 remains sensitive, resulting in a shift in molecular weight (arrowhead, lane 12). Treatment of the protoplasts with 20 μg/mL BFA to redistribute the Golgi enzymes responsible for glycan modification to the ER resulted in the glycan on TMcCCASP^DXE1 becoming resistant to the endoglycosidase, in the same manner as spNGFP-HDEL (lanes 6 and 14). UT, untransformed sample.