FIGURE 7.

Localization of transiently expressed EmGFP-tagged SYT1, SYT5, and SYT1 proteins mutated at the C₂B calcium-binding site in protoplasts. A, structural model of the SYT1 C₂B domain. Diagrammatic representation of the C₂B domain of SYT1 based on the prediction of putative β-strands in the C2 domains of eukaryotic synaptotagmin-related proteins by Jiménez and Davletov (16). Graduated-blue colored strips indicate putative β-strands. The strands are labeled in numerical order from the N terminus to the C terminus. B, comparison of the calcium-binding sites at Loop 1 and Loop 3. Amino acid sequences of SYT1, SYT2, SYT4, SYT5, and SYT1 mutants are aligned. Asterisks indicate amino acid residues of the conserved calcium-binding motifs. C, projections of optical sections of protoplasts. Transfected protoplasts were observed with confocal fluorescence microscopy. The optical sections were reconstructed as a projection image. GFP and autofluorescence of chlorophyll are green and red, respectively. D, colocalization analysis of SYT1 mutants containing both Loop 1* and Loop 3* in protoplasts isolated from the leaves of Golgi-labeled transgenic plants. GFP and CFP are green and magenta, respectively. BF, bright field; GFP, GFP fluorescence; CP, autofluorescence of chlorophyll; Golgi, CFP fluorescence of CFP-labeled Golgi marker proteins; Merged, merged image of GFP with CP or CFP. Bars indicate 10 μm.