Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - KAT1 K⁺ channel is associated with a moderately detergent-resistant membrane fraction. Western Blot analysis of KAT1 K⁺ channel (haKAT1:GFP) after expression in tobacco leaves. SDS-PAGE (10 μg total protein/lane) of microsomal membranes after partitioning in 1% Triton-X100 at detergent:protein ratios (w/w) of 10:1 and 5:1 (Mongrand, et al. 2004). After blotting, nitrocellulose filters were probed with polyclonal antibody to GFP and visualised by chemiluminescence. The microsomal membrane fractions used will have included endomembranes which may have affected the apparent distribution of the K⁺ channel protein between detergent-resistant (DR) and -senstive (DS) fractions. Note the presence of the channel protein in the detergent-resistant fraction at the lower, but not at the higher detergent:protein ratio.
• Supplemental Figure 2 - Confocal dual-labelling analysis for the images in Fig. 10 and Supplementary Movie 5 of a protoplast from tobacco leaf tissue previously transfected with haKAT1:GFP together with either the Sp2 fragment of NtSyp121. Protoplasts were bound with monoclonal antibody to hemaglutinin covalently tagged with Alexafluor594 (Alexa594-αHA) for 5 min prior to imaging and are shown here in tangential surface views as (left to right): brightfield, Alexa594, GFP and chloroplast fluorescence. Note the absence of discrete boundaries to, and apparent merging between K⁺ channel domains. Imaging parameters as in Fig. 6. Scale bars, 5 μm.
• Supplemental Movie 1 - haKAT1:paGFP is non-mobile at the cell periphery. Movie constructed from brightfield and GFP (grayscale) confocal fluorescence images from one experiment with tobacco epidermal cells expressing the fusion construct. Corresponding still image frames will be found in Fig. 3. Images were collected at 10-s intervals before and after 4-μs photoactivation with 351/364 nm light within the area circled. Time in seconds relative to photoactivation as indicated (bottom right).
• Supplemental Movie 2 - Comparative analysis of fluorophore mobilities by fluorescence recovery after photobleaching (FRAP) with the H⁺-ATPase PMA2:GFP. Movie constructed from brightfield and GFP (grayscale) confocal fluorescence images from one experiment with tobacco epidermal cells expressing the fusion construct. Images were collected at 10-s intervals before and after photobleaching with 458/488 nm light within the areas circled. Time in seconds relative to photoactivation as indicated (bottom right). Corresponding still image frames will be found in Fig. 4A.
• Supplemental Movie 3 - Comparative analysis of fluorophore mobilities by fluorescence recovery after photobleaching (FRAP) with GFP:HDEL that labels the endoplasmic reticulum. Movie constructed from brightfield and GFP (grayscale) confocal fluorescence images from one experiment with tobacco epidermal cells expressing the fusion construct. Images were collected at 10-s intervals before and after photobleaching with 458/488 nm light within the areas circled. Time in seconds relative to photoactivation as indicated (bottom right). Corresponding still image frames will be found in Fig. 4B.
• Supplemental Movie 4 - haKAT1::paGFP is mobile at the epidermal cell periphery when co-expressed with the Sp2 fragment of tobacco SYP121. Movie constructed from brightfield and GFP (grayscale) confocal fluorescence images from one experiment with tobacco epidermal cells expressing the fusion construct and Sp2 fragment. Images were collected at 10-s intervals. Time in seconds relative to photoactivation as indicated (bottom right). Corresponding still image frames will be found in Fig. 9.
• Supplemental Movie 5 - haKAT1:GFP is mobile at the plasma membrane when co-expressed with the Sp2 fragment of tobacco SYP121. Movie constructed from brightfield and Alexa594 (grayscale) confocal fluorescence images from one experiment with tobacco epidermal cells expressing the fusion construct and Sp2 fragment after pretreatment with monoclonal antibody to hemaglutinin covalently tagged with Alexafluor594 (Alexa594-αHA) for 5 min prior to imaging. Images were collected at 2-s intervals. Time in seconds relative to photobleaching as indicated (bottom right) and area of photobleach is circled in the frame immediately beforehand. Corresponding still image frames will be found in Fig. 10.