Supplemental Data

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• Supplemental Figure 1 - Supplemental Figure 1. Localization of GFP-BS14a in cycloheximide-treated tobacco BY-2 cells. Cells were transformed with the fusion protein GFP-BS14a consisting of GFP fused to the Arabidopsis SNARE BS14a. At 4 h after bombardment, cells were either fixed in formaldehyde or incubated for an additional 1 h (5 h total) in the presence or absence of cycloheximide, followed by formaldehyde fixation. Transformed cells were also stained with concanavalin A conjugated to Alexa 594. Note that while the majority of nascent GFP-BS14a localized to ER at 4 h post-bombardment and at 5 h post-bombardment in the absence of cycloheximide, GFP-BS14a accumulated in the Golgi at 5 h post-bombardment in the presence of cycloheximide, consistent with its sorting from ER to Golgi (Uemura et al., 2004). The DIC image corresponds to the same cell transformed with GFP-BS14a and incubated with cycloheximide for 5 h. Bar = 10 μm.
• Supplemental Figure 2 - Supplemental Figure 2. p33-K₅R₆K₁₁K₁₂ΔG co-localizes with CAT-APX in aggregated peroxisomes, but not in pER, in tobacco BY-2 cells. Cells were co-transformed with p33-K₅R₆K₁₁K₁₂ΔG and CAT-APX, incubated for 4 h, then fixed in formaldehyde and processed for immunofluorescence microscopy. Arrowheads indicate obvious co-localizations of p33-K₅R₆K₁₁K₁₂ΔG and CAT-APX in aggregated peroxisomes. Bar = 10 μm.
• Supplemental Figure 3 - Supplemental Figure 3. p33 contains an arginine-based ER retrieval motif. (A) Conserved amino acid substitutions of the putative arginine-based ER retrieval motif in p33 (-K₅R₆-) preserves its sorting to pER in tobacco BY-2 cells. Cells transformed with a modified version of p33 possessing a prototypic arginine-based ER retrieval motif (-R₅R₆-; p33-K₅ΔR) were formaldehyde fixed either at 4 h(a) and (b) or 24 h (c) and (d) after bombardment and processed for immunofluorescence microscopy. Solid arrowheads in (a) and(b) and (c) and (d) indicate obvious co-localizations of expressed p33-K₅ΔR and endogenous catalase in small globular (aggregated) peroxisomes. Localization of p33-K₅ΔR also to pER is evident in (c); compare with the localization of wild-type p33 at similar time points in Fig. 3A. Bar in (a) = 10 μm. (B) Schematic representation of α-glucosidase I-GFP chimeric proteins. Chimeric proteins are drawn to scale and consist of either the N-terminal 100 amino acid residues from Arabidopsis α-glucosidase I including the protein’s arginine-based ER retrieval motif (underlined, MTGASRR-) or a modified version thereof, including MTGASAA- and METIKRM-. Also shown is the α-glucosidase I single putative membrane-spanning domain (residues 52-69) and the GFP (residues 101-337). Selected N-terminal sequences shown are indicated by single letter code. (C) The putative arginine-based ER retrieval motif in p33 preserves the ER localization of α-glucosidase I in BY-2 cells. BY-2 cells transformed with either α-glucosidase I-GFP (a), α-glucosidase I-GFP R₆R₇ΔA (c), or METIKRM-α-glucosidase I-GFP (e) (illustrated in [B]) were formaldehyde fixed at 24 h after bombardment and processed for immunofluorescence microscopy. Transformed cells were also stained with concanavalin A conjugated to Alexa 594 (b), (d) and (f). Solid arrowheads in (a) and(b) and (e) and (f) indicate obvious co-localizations. Note that α-glucosidase I-GFP R₆R₇ΔA completely (mis)localized to the Golgi; obvious non-colocalizations between expressed α-glucosidase I-GFP R₆R₇ΔA (c) and concanavalin A-stained ER (d) are indicated with open arrowheads. Bar in (a) = 10 μm.