Figure 4.

All family members are localized to chloroplasts in vivo. A, AtACHT proteins fused with GFP at their C terminus were transiently expressed in Arabidopsis protoplasts. Images of the GFP-fused AtACHT (AtACHT:GFP) fluorescence and the chlorophyll autofluorescence (CHLR) were combined (MERGE) to illustrate the relative localization of the GFP fluorescence and the chloroplasts. Markers for chloroplast (Chpst), mitochondria (Mito), ER, and cytoplasm (Cyto) were visualized as well. B, Transgenic plants, each expressing AtACHT1:YFP or AtACHT4a:YFP, were compared to plants expressing cytoplasmic YFP under the control of 35S promoter. The fluorescence images corroborated that both AtACHT1 and AtACHT4a are targeted to chloroplasts in planta. C, Subfractionation of intact chloroplasts from transgenic plants expressing AtACHT fused with HA₃ affinity tag into stroma and thylakoid membranes demonstrates the suborganellar localization of AtACHT1 and AtACHT4a by immunoblot using anti-HA antibody. The stromal large subunit of Rubisco (LSU) and the membranal D1 protein were blotted for quality assessment of the fractionation procedure.