Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1
• Supplemental Table 2
• Supplemental Figure 1 - Supplemental Figure 1. Multiple alignments of all VTE5 homologues and dolichol kinases used to generate the tree shown in Fig. 5. Multiple alignments were visualized and edited using GeneDoc (Nicholas et al., 1997). The residues are shaded using conserved residue shading mode set to level 4 using default settings. Similar amino acid residues conserved in all columns are shaded dark. Amino acids with 100%, 80%, and 60% conservation are shaded as white letters on black background, white letters on dark grey background, and black letters on light grey background, respectively. Amino terminal non-conserved amino acids encoding putative chloroplast target sequences were deleted from the alignment. Numbers above the sequence refer to amino acid positions within the Arabidopsis VTE5 sequence. (red down arrow): location of vte5-1 mutation, which is predicted to cause truncation of the remaining amino acid residues. Reference numbers: Synechocystis vte5, nucleotides 3452461 – 3453162, Accession # gi|47118304. Abbreviations: Aa, Aquifex aeolicus VF5; Af, Archaeoglobus fulgidus; At, Arabidopsis thaliana; Av, Anabaena variabilis; Ca, Chloroflexus aurantiacus; Ct, Chlorobium tepidum; Gm, Glycine max; Np, Nostoc punctiforme; Os, Oryza sativa; Pa, Pyrococcus abyssi; Sc, Saccharomyces cerevisiae; Syn, Synechocystis sp. PCC 6803; Te, Thermosynechococcus elongatus; Zm, Zea mays.
• Supplemental Figure 2 - Supplemental Figure 2. Total and extracted ion chromatograms from LC-MS analysis of (A) standard mix and (B) E. coli cell extract. The GGDP and PDP standard mixture, which contained small amounts of GGMP and PMP, respectively, was used at a concentration of 1 pmol/μL. Panel A1 represents the total ion chromatogram of the standard mix and panels A2 through A5 represent the extracted ion chromatograms (XIC) for GGDP, GGMP, PDP, and PMP from the standard mix, respectively. The total ion chromatogram of E. coli extract shown in panel B1 was obtained from E. coli (pET30b-Arabidopsis VTE5) which had been incubated with phytol. Panels B2 through B5 represent the extracted ion chromatograms of this E. coli sample for GGDP, GGMP, PDP, and PMP, respectively. The combination of retention time and m/z was unique for each compound and could be used to identify the presence of each in the samples. The peak areas were used for relative quantitation.
• Supplemental Figure 3 - Supplemental Figure 3. LC-MS/MS verification of isoprenoidmonophosphate formation in E. coli. E. coli (pET30b-Arabidopsis VTE5) was incubated for 3 h with free phytol and extracted as described in the Methods section. Panels A through C show the total ion chromatogram (A), MS/MS product ion spectrum of parent mass 369.1 (GGMP) taken at 2.79 min (B), and the MS/MS product ion spectrum of parent mass 375.1 (PMP) taken at 2.94 min (C). Parent ion masses (circled in green) were fragmented in the mass spectrometer using CID to generate daughter ions. Two prominent masses were observed in both spectra (circled in orange). Both of these masses confirm the presence of phosphate in the parent compounds: m/z 96.9 = H₂PO₄- and m/z 78.9 = PO₃-.