Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - VHA-a1-GFP: Expression levels and integration into the V-ATPase complex. (A) RT-PCR on total RNA of transgenic lines expressing VHA-a1-GFP. Specific primer sets were used for VHA-a1(lanes 1-3) and VHA-a1-GFP (lanes 4-6). Notice that in transgenic line 9-2 transcript levels of VHA-a1 and of VHA-a1-GFP are comparable. (B) Coimmunoprecipitation of V1-subunit VHA-A with VHA-a1-GFP. Total protein extracts of Col-0 (lanes 4-6) and Col-0 VHA-a1-GFP (lanes 1-3, 7-9) were used for immunoprecipitation with a specific antibody against GFP (lanes 2, 3, 5, 6) and HA (lanes 8, 9). For each sample, the abundance of the V1-subunit VHA-A was analyzed in the supernatant before (S1) and after precipitation (S2), as well as in the pellet (P) by Western Blot analysis using the specific antibody against VHA-A. Comparison of lanes 1 and 2 shows that a substantial fraction of VHA-A is present in complexes with VHA-a1-GFP.
• Supplemental Figure 2 - Ubiquitous expression of VHA-a isoforms. Expression of VHA-a1-GFP (A-D), VHA-a2-GFP (E-H) and VHA-a3-GFP (I-L) in cotyledons (A,E,I), hypocotyls (B,F,J), roots (C,G,K) and root tips (D,H,L) of etiolated seedlings, Inset in L shows that VHA-a3-GFP is found at the tonoplast.
• Supplemental Figure 3 - Colocalization of SYP41-GFP and SYP41-mRFP. (A) to (C) Protoplasts transiently expressing SYP41-GFP and SYP41-mRFP. Images are maximum projections derived from Z-scans of individual protoplasts. Shown are the separately recorded GFP (A) and RFP (B) channels as well as an overlay of both channels (C).
• Supplemental Figure 4 - Linescan analysis of colocalization. Line scans displaying pixel intensities along arbitrary lines in simultaneously obtained CLSM-pictures of (A) VHA-a1-GFP + ARA7-mRFP, (B) VHA-a1-GFP + SYP41-mRFP and (C) VHA-a1-GFP + FM4-64 (6 minutes)
• Supplemental Figure 5 - Transport of FM4-64 to the tonoplast in suspension cells. (A) and (B) Suspension culture cells untreated (A) or pretreated with ConcA three hours after FM4-64 staining. Note the absence of tonoplast staining in B.
• Supplemental Table 1