Figure 4

Mutational analysis of amino acids required for StAR START domain activity in yeast assay. (A) Structural alignment of START domains from MLN64, StAR, STARD4 and PCTP with MLN64 as a reference. Consensus α-helices and β-sheets are displayed with ESPript/ENDscript. Amino acids targeted for site-directed mutagenesis of StAR START are indicated in yellow. Ligand contact points (green), as derived from the PCTP-PtCho co-crystal [14], are shown for PCTP. (B) Site-directed mutagenesis of the START domain from mammalian StAR. Activities are indicated for GSV constructs that contain missense mutations in the START domain. The M143R;N147D and R181L mutants display wild-type and slightly elevated activities, respectively. L270M renders partial activity, while the other five mutants exhibit low or no activity. Uninduced GEV served as the control. Error bars indicate standard deviations for two independent transformants in four trials. (C-F) Trichomes are shown from second leaves of Arabidopsis seedlings. Scale bar: 100 μm. (C) Wild-type (WT), (D) gl2 mutant and gl2 lines transformed with (E) ProGL2:EYFP:GL2-StAR-START and (F) ProGL2:EYFP:GL2-StAR-D182L-START, in which the START domain of StAR contains the D182L missense mutation. Live imaging of leaf trichomes indicates nuclear localization of (H) EYFP:GL2-StAR-START and (J) EYFP:GL2-StAR-D182L-START (white arrows). (G, I) Light (green, chlorophyll) and (H, J) matching fluorescence (red, chlorophyll). Scale bar: 100 μm. (K) Quantification of trichomes on first leaves. Error bars indicate standard deviations for n = 20. The asterisk marks a significant difference between EYFP:GL2-StAR-START and EYFP:GL2-StAR-D182L-START (two-tailed t-test, P < 0.00001). EYFP, enhanced yellow fluorescent protein; GEV, Gal4 DNA binding domain:estrogen receptor:VP16 activation domain; GL2, Glabra2; MLN64, Metastatic Lymph Node 64; PCTP, phosphatidylcholine transfer protein; PtCho, phosphatidylcholine; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer; WT, wild type.