Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. GONST1-GFP is Transported to the Golgi and to Smaller, non-Golgi Structures To verify the non-Golgi localisation of the smaller GONST1-GFP structures, sequential images of cells expressing GONST1-GFP and ERD2-YFP were taken through the cell cortex at steps of 0.44 μm apart. Each step is shown as a separate image. Note that the smaller GONST1-GFP structures do not colocalise with ERD2-YFP. Note also that some of the non-Golgi structures (marked by arrowheads) move in the Z plane as well as the XY plane and therefore appear in several consecutive frames. Scale bar = 2 μm.
• Supplemental Figure 2 - Supplemental Figure 2. GONST1-GFP^DXE is Able to Be Transported to the Golgi and to Smaller, non-Golgi Structures Sequential images of cells expressing GONST1-GFP^DXE and ERD2-YFP were taken through the cell cortex at steps of 0.44 μm apart. Each step is shown as a separate image. GONST1^DXEE labels ER, Golgi apparatus and smaller, non-Golgi structures (marked by arrowheads), which do not colocalise with ERD2-YFP. Scale bar = 2 μm.
• Supplemental Figure 3 - Supplemental Figure 3. GONST1-GFPEXD Labels the Golgi and Smaller, non-Golgi Structures Sequential images of cells expressing GONST1-GFP^EXD and ERD2-YFP were taken through the cell cortex at steps of 0.44 μm apart. Each step is shown as a separate image. GONST1^EXDD labels only the Golgi apparatus and smaller, non-Golgi structures (marked by arrowheads). Scale bar = 2 μm.
• Supplemental Figure 4 - Supplemental Figure 4. GONST1-GFP^Δcyt is partially Retained in the ER but is also Transported to the Golgi apparatus and Small non-Golgi structures Sequential images of cells expressing GONST1-GFP^Δcytt and ERD2-YFP were taken through the cell cortex at steps of 0.44 μm apart. Each step is shown as a separate image. Note the labelling of ER, Golgi apparatus and smaller, non-Golgi structures (marked by arrowheads) by GONST1^Δcyt, whereas ERD2-YFP labels only ER and Golgi apparatus. Scale bar = 2 μm.
• Supplemental Figure 5 - Supplemental Figure 5. GONST1^DXE-GFP is Transported to the Same Small Dots as Wild-Type GONST1 (A) GONST1^DXE-GFP labels the ER, the Golgi apparatus and small punctate structures of unknown nature (arrowhead). Co-expression of GONST1-YFP (B) confirms that these small structures are the same as those labelled by wild-type GONST (arrowhead). (C) The co-localisation of the two proteins on Golgi and smaller dots can clearly be seen in the merged image, although ER staining is only seen for GONST1^DXE-GFP. Size bar = 5 μm.
• Supplemental Figure 6 - Supplemental Figure 6. The EXD Motif in the N-terminus of GONST1 Does Not Regulate its ER Export (A) GONST1^EXD-GFP labels punctate structures of different sizes when expressed in tobacco leaf epidermal cells. (B-D) The larger structures labelled by GONST1^EXD-GFP (B) were shown to be Golgi bodies through co-expression of ERD2-YFP ((C), arrowhead), although the smaller structures did not colocalise with ERD2-YFP (arrow). (D) Merged image of (B) and (C). Size bar = 5 μm.
• Supplemental Figure 7 - Supplemental Figure 7. Mutation of the EXD Motif in CASP has No Effect on its Transport Out of the ER (A) YFP-CASP^EXD labels punctate structures when expressed in tobacco leaf epidermal cells. (B-D) Co-expression of ERD2-GFP(C) with YFP-CASP^EXD (B) confirms that these structures are Golgi bodies (D). Size bar = 5μm.
• Supplemental Figure 8 - Supplemental Figure 8. The EXD Motif Does Not Function as an Export Signal in TMcCCASP (A) TMcCCASP^EXD labels punctate structures when expressed in tobacco leaf epidermal cells. This distribution is identical to that of TMcCCASP (B). Size bar = 5 μm.
• Supplemental Figure 9 - Supplemental Figure 9. Expression of ER-Retained Mutants of GONST1 Does Not Alter the Golgi Localisation of ST-mRFP (A-C) GONST1-GFP and ST-mRFP (Saint-Jore et al., 2002; Renna et al., 2005) colocalise at the Golgi apparatus. (D-F) GONST1^DXE-GFP labels both the ER and Golgi apparatus (D), but ST-mRFP labels only the Golgi (E). If expression of the mutant proteins altered ER-Golgi transport of the Golgi marker, a redistribution of ST-mRFP into the ER would occur. (G-I) GONST1^δcyt-GFP colocalises with ST-mRFP at the Golgi apparatus (I), but not at the ER. The localisation of ST-mRFP is unaffected by co-expression of the mutant protein (H). (J-L)To show the effects of disrupted ER-Golgi transport on the localisation of ST-mRFP, we treated cells co-expressing GONST1-GFP and ST-mRFP with 100 μg/ƒÝl BFA for one hour. This resulted in the redistribution of most of the Golgi-localised proteins to the ER. Size bars = 5 μm.
• Supplemental Figure 10 - Supplemental Figure 10. Expression of ER-Retained Mutants of CASP and TMcCCASP Does Not Alter the Golgi Localisation of ST-mRFP (A-C) GFP-CASP and ST-mRFP colocalise at the Golgi apparatus. (D-F) Although GFP-CASP^DXE1 localises at both the ER and Golgi (D), its expression does not affect the localisation of ST-mRFP (E). (G-I) TMcCCASP colocalises with ST-mRFP at the Golgi apparatus (I), but only TMcCCASP exhibits faint ER staining(G). Fluorescence from an out-of-plane chloroplast is faintly visible in the magenta channel (arrowhead). (J-L) TMcCCASP^DXE1 is found only in the ER (J), while co-expressed ST-mRFP localises only at the Golgi apparatus (K). Size bars = 5 μm.