Figure 7.

Stage-specific farnesylation and subcellular localization of AtNAP1;1 directs its functions. A, Western-blot analysis of AtNAP1 proteins during wild-type and era1-1 primary leaf development. Samples were collected after 9, 14, 19, and 25 d for wild type (lanes 1–4) and 11, 16, 21, and 27 d for era1-1 (lanes 5–8). The difference of harvesting time originates from a 2-d delay in era1-1 germination. Twenty-five micrograms of total protein was loaded per well and separated by SDS-PAGE in 7.5% gel followed by western blotting. Membrane was probed with polyclonal AtNAP1 antibody. B, In vivo prenylation assay of AtNAP1;1 during leaf development. The 9- and 15-d-old wild-type (lane 1), AtNAP1;1-OE (lane 2), and AtNAP1;1^C369S-OE (lane 3) first leaves were labeled with [³H]mevalonic acid, and protein extracts were subjected to immunoblot analysis and fluorography. Fluorography was carried out for 3 weeks. C, Subcellular fractionation of 9- and 15-d-old wild-type, AtNAP1;1-OE, and AtNAP1;1^C369S-OE first leaf protein extracts. Twenty micrograms of total protein (T) and equivalent volumes of cytoplasmic (C) and nuclear (N) fractions were loaded. D, Subcellular fractionation of 9- and 15-d-old wild-type and 11- and 17-d-old era1-1 primary leaf protein extracts. The experiment was carried out as described for C. As control, 50 μg of total protein and equivalent volumes of the two other fractions were used for detection using the antibody raised against the MSI nuclear protein.