Figure 4.

Subcellular localization of various Arabidopsis SEN proteins and human spartin, and their corresponding SDs. N. benthamiana leaves were transiently transformed (via Agrobacterium infiltration) with either full-length or the C-terminal portion, including their SD, of Arabidopsis SENA2, SENA3, SENB1, SENB2, or human spartin, and tagged at their C-termini with mCherry. Three days post-infiltration, leaves were stained with the neutral lipid dye BODIPY and imaged using CLSM. Alternatively, leaves were co-transformed with either SENB2-SD-mCherry or SENB1-SD-mCherry and the mitochondrial marker protein GFP-mito. (A) Schematic representations of the full-length and/or C-terminal SD-containing portion of each Arabidopsis SENA/B protein and human spartin, and their corresponding subcellular localization(s) [i.e., cytosol, lipid droplet (LD), mitochondria (mito)]. The SD in each protein is based on information obtained from the InterPro domain database. The numbers next to the name of each SD construct denote the amino acid residues that were fused to mCherry. The portion of each protein representing the SD is depicted in the illustrations as a light green box, while other regions of the SENA, SENB, and spartin proteins are depicted as gray, dark green, and blue boxes, respectively. Note that the C-terminal-appended mCherry moiety is not depicted in the illustrations or construct names. Representative CLSM images (z-sections) of mCherry-tagged full-length or truncated SEN-family proteins (B) or spartin (C), along with the corresponding BODIPY-stained LDs or co-expressed GFP-mito in the same cell, and as indicated by panel labels. Also shown is the corresponding merged image. Arrowheads indicate examples of SENA/B or spartin fusion proteins that localized to BODIPY-stained LDs or GFP-mito-labeled mitochondria. Scale bars in (B,C) = 10 μm.