Article Figures & Data
Figures
- Fig. 1.
Phylogenetic analysis of selected syntaxin-type t-SNAREs from human (Hs), yeast (Sc), and Arabidopsis (At). Full-length protein sequences were acquired from GenBank (see “Materials and Methods” for accession numbers), aligned, and a phylogenetic tree prepared using the Jotun Hein algorithm of the MEGALIGN program in the DNASTAR package. Below the tree is a scale relating the distance between the sequences. All branches have been truncated (vertical lines) to improve display. See text for details.
- Fig. 2.
Preparation of antisera specific to AtPEP12p or AtVAM3p. A, Antibodies raised against C- or N-terminal epitopes of AtPEP12p or against the cytoplasmic domain of AtVAM3p recognize bands of similar size (approximately 36 kD) following SDS-PAGE of Arabidopsis microsomal extracts. B, Affinity-purified antisera to each t-SNARE do not cross-react. Extracts from yeast cells expressing the cDNA forAtPEP12 (lanes 1, 3, and 5) or AtVAM3(lanes 2, 4, and 6) were separated by SDS-PAGE, then probed with affinity-purified antisera to either C-terminal-specific AtPEP12p, N-terminal-specific AtPEP12p, or AtVAM3p. C, Expression of epitope-tagged t-SNAREs in transgenic Arabidopsis. Microsomal extracts from wild-type plants (lanes 1, 4, and 7), plants expressingT7-AtPEP12 (lanes 2, 5, and 8), and plants expressingT7-AtVAM3 (lanes 3, 6, and 9) were separated by SDS-PAGE, and then probed with T7 monoclonal antibodies or with affinity-purified antisera to either C-terminal-specific AtPEP12p or AtVAM3p.
- Fig. 3.
AtVAM3p and AtVTI1a interact in vivo. A detergent extract of wild-type (wt) or transgenic Arabidopsis plants expressingT7-AtVTI1a was immunoprecipitated with T7-monoclonal antibodies. Shown are aliquots of the total extract (T), the flowthrough (FT), and the elution (E) from the immunoprecipitation separated by SDS-PAGE, then probed with AtPEP12p-specific or AtVAM3p-specific antiserum.
- Fig. 4.
AtVAM3p co-fractionates with PVC markers in Suc density gradients. A, Microsomal extracts of wild-type Arabidopsis plants were separated on Suc density gradients. Twenty-four fractions were taken, TCA precipitated, resuspended in SDS sample buffer, and equal volumes separated by SDS-PAGE. Strips of these blots were probed with antisera specific to: H+-pyrophosphatase (PPase), AtELP, C-terminal specific AtPEP12p, or AtVAM3p. B, These blots were digitized on a flat-bed scanner, and densitometry was performed with imaging software. Shown is a quantification of each fraction (relative to the total amount of each protein loaded onto the gradient) for H+-pyrophosphatase (PPase, ⋄), AtELP (■), AtPEP12p (▴), and AtVAM3p (●). C, The density profile of this gradient was determined by refractometry and is virtually linear. D, Microsomal extracts of plants either expressing T7-AtPEP12 (■) orT7-AtVAM3 (▴) were separated on Suc density gradients as described for A, and quantified as described in B. The density profile of each gradient was similar to that shown in C.
- Fig. 5.
AtVAM3p localizes to the PVC in Arabidopsis root cells. Cryosections of the root tip of transgenic Arabidopsis seedlings expressing either T7-AtPEP12 or T7-AtVAM3were prepared and stained with T7-monoclonal or specific antisera to AtPEP12p (C-terminal specific) or AtVAM3p. In all cases, AtVAM3p is represented by 10-nm gold particles (arrows), while AtPEP12p is represented by 5-nm gold particles (arrowheads). A, Cryosections ofT7-AtVAM3-expressing transgenic plants stained with T7-monoclonal antibodies detected with 10-nm gold. B, Cryosections ofT7-AtVAM3-expressing transgenic plants stained with no antisera followed by 10-nm gold. C, Cryosections ofT7-AtVAM3-expressing transgenic plants stained with T7-monoclonal antibodies detected with 10-nm gold and with affinity-purified AtPEP12p antiserum (C-terminal specific) detected with 5-nm gold. D and E, Cryosections ofT7-AtPEP12-expressing transgenic plants stained with T7-monoclonal antibodies detected with 5-nm gold and with affinity-purified AtVAM3p antiserum detected with 10-nm gold. F, Cryosections of T7-AtPEP12-expressing transgenic plants stained with no antibodies followed by 10-nm gold and preimmune serum for AtPEP12p detected with 5-nm gold. Bar = 500 nm.
