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(a) 1 2 3 205 kD 116 kD 97 kD 66 kD 29 kD n m o lG 1 P /h /m l 8 (b) phoA 6 phoB 4 2 0 10 20 30 40 50 te m p e ra tu re (°C ) 60 8 nmolG1P/h/ml (c) phoA phoB 6 4 2 0 4 5 6 7 8 pH 9 10 11 12 Supplemental Fig. 1: Purification of PhoA and PhoB proteins. (a) One microgram of each purified proteins were loaded on a 7,5 % acrylamide SDS-PAGE. The gel was then stained with Coomassie Brilliant Blue. Lane 1 : molecular weight marker, Lanes 2 and lanes 3 correspond respectively to the purified PhoA and PhoB phosphorylases. (b) pH response of purified PhoA (open circles) and PhoB (solid circles) enzymes adjusted to equal enzyme activities (at pH7 and 30°C) using Tris-maleate-glycine universal buffer. The assay was performed at 30°C in the presence of 10 mg.mL-1 rabbit liver glycogen and 10 mM orthophosphate (c) Temperature curves of purified PhoA (open circles) and PhoB (solid circles) enzymes adjusted to equal enzyme activities activities (at pH 7 and 30°C). The assay was performed at pH 7 in the presence of 10 mg.mL-1 rabbit liver glycogen and 10 mM orthophosphate 1 (a) MAYRQLQGTNRGASAGAPVAYSRPMQGRVGRSALRVQAVAEAERPTAAKSSGSAEPVTTDITSKL KYLFGRNGDYTNADAYQGTAWSVREKLIDSFNKTHEHWKKEDPKFIYYLSAEFLMGRSLTNTVYN LGLEGEYGNALREMGYHMEKVADAERDAALGNGGLGRLAACFLDSMATLDLPGWGYGIRYKYGMF KQGLKDGYQVEMPDIWLTKGNPWEVRRDDVKFEVGFGGRVERKKVNGKEMTVWTPSEKVIAQAYD NPIPGYATPTTSNLRLWDAVPVHEFDLSAFNAGDYDRAMLERERAEGISAVLYPNDSTPEGKELR LKQQYFFVCASLQDVMSRFRAVHGANWEALPEKACFQLNDTHPTIAVAELMRLLVDVEGLEWDAA WTITTKCLNYTNHTVMPEALEKWPVKVMAKMLPRHMEIIEVINEGWTKWLGVHLKDLKSEERAKK IAAMSIIHANPWNADEMLVNMAYLAVVGSSAVNGVAAIHSNIVKDEILNDFYEIFPSKFQNKTNG VTPRRWLAWCNPELAQLITEALGSSEWINDTEKLAGLRAFASDPAFQAKWAAVKKAKKAKLAELI KKIHGDDVNQNALFDIQIKRIHEYKRQYLNVLSIIWRYKQLKKMTPEQRKASAVPRVCVIGGKAA SAYDMAKRIIRLVTAVGEVINKDPETKDYLRLYFLPDYNVTLAETIIPAAELSQHISTAGTEASG TSNMKFQMNGCLIMGTWDGANIEIAEETGVENVFVFGVRAEEINQLRKDRKNFKTDPRWDELMKD IEGGMFGDKDYFKPLVDSVNNMKVGNDWFLLANDFAGYLAAQEEVDATYKDQAEWLRRSIMYTAG SGKFSSDRTIREYAEDIWHVKPARPSA (b) MQLASRALAASAAALSAPRAAKCAVPAPVAVSASRSKAVAPTRRSQGPGRQVSVKVAAPESPASS GEVIVNFDNTTDSGYTVISVQANNKPGLLTSITALFRDLGVDVGKAVVEGDEDRINDKFYVRSLS GGKLSEDKAADCVKALDVLLRSKPTGTEATRPKFENTAATGGTGKARLYTLMDTYMKNDVLSIQE DIVNHVEYTLARSRVNFDNFEAYQATSFSLRDRLIERWNDTQTWFKEKDPKRVYYLSMEFLMGRS LLNTLYNLDIKESYQEALAELGYDLETLADLERDAALGNGGLGRLAACFLDSMATLNLPAWGYGI RYQYGMFRQTIQNGFQHEQPDYWLTFGNPWEIERLIVSYPIKFYGHVSVVNEDGRQLFRWNAGET VTAVAYDNPIPGFGTRNCINLRLWAAKPSKEFDLEAFNTGDYVAAILSKQRAETLSSVLYPDDRT YEGKELRLKQQHFFVSATIQDCVRRYRDAHPNDWEQFPEKVAFQLNDTHPTIAVAELMRVLMDDH KLGWTKSWDICNKVFAFTNHTVLPEALERWPVALIEKLLPRHMQIIYDINWRFLQTVRNKFGDDW ERISRMSVIEEQPNGEKMVRMAFMAVVASHTVNGVAAIHSEIIKETIFKDFYELWPNKFQNKTNG VTQRRWLAFCNPPLRQLITKKLGNDDWTLHLDNLRELRKYANDPEFQTEWRGVKSEAKKKAAALI HRLTGVRVSTDAMFDIQIKRIHEYKRQLLNVLGIIYRYDQIKKMTPQQRKSVVPRVCVIGGKAAP GYEMAKRIIKLICAVGDKINQDPDMGDLLKLVFLPDYNVSSAEVIIPATELSQHISTAGTEASGT SNMKFTMNGSLIIGTLDGANVEIAEEIGDENIFIFGAKAHEVARLRAERRNLHVDERFNHVVNMI RTGHFGWEDYFGPVVDAITTGGDYYLVANDFPGYLETQFRADEVYKNQTEWTRMSIMATAGGGKF STDRTIAEYARDIWHAEPCQVPQPEAKSKSKPASS Supplemental figure 2 Mass spectrometric analysis of purified PhoA and PhoB proteins. The proteins were digested “in gel” with trypsin before being analyzed by various mass spectrometric techniques. The sequences of PhoA (a) and PhoB (b) are displayed. The expected molecular weights of the peptides printed in colour could be matched with a peptide mass fingerprint obtained by MALDI-TOF MS. The sequence stretches displayed in red were confirmed by sequencing with ESI-Q-TOF tandem mass spectrometry. For Pho A (a) the peptide displayed in bold faced red was identified as the Nterminus of the protein: The peptide is not preceded by a lysine or an arginine, (and, therefore, it can not originate from the tryptic digestion). Furthermore, the N-terminal alanine was shown to be acetylated. The mature Pho A sequence therefore, starts with AcVAEAERPTAAKSS. Unfortunately, the N-terminaus of Pho B (b) could not be identified. 2 0.8 (a) 0.147 0.132 -Log (Rf) 0.6 0.140 0.115 0.097 0.4 0.043 0.2 0 7 8 9 10 Acrylamide % 250 MW (kD) (b) 200 150 100 0.00 0.05 0.10 0.15 0.20 Slope Supplemental Fig. 3.: PhoA and PhoB are dimerics in their native states. 3 The dimeric nature of both PhoA and PhoB in native conditions were determined by comparing the mobility of each purified protein on native acrylamide gels containing different acylamide concentrations (7, 8, 9, 10 % monomer). These mobilities were compared to those of purified proteins of known molecular weights including plastidial potato phosphorylase, 208 kD ; cytosolic potato phosphorylase, 194 kD; Escherichia coli alcohol dehydrogenase, 150 kD; and Sweet potato -amylase, 200kD. The mobility of each protein is shown in (a) for the 4 acrylamide concentrations used. The slope obtained for PhoA and PhoB were then estimated on a graph built with the slopes calculated from the proteins with known molecular weights (b). 4 Starch amounts (mg) 2 1.5 1 0.5 0 0 24 48 72 96 Time (hours) Supplemental Fig 4: Kinetics of starch deposition of wild-type and sta4 mutant strains during nitrogen starvation. Late log-phase culture of two wild-type and and two sta4-1 mutant progeny from the same tetrad were used to inoculate one liter of nitrogen starved medium at a density of 106 cells per mL (T=0). Sampling was performed during 80 hours. For each time point, 50 mL of cultures were used to purify and assay starch. The results are displayed as mg of starch in the total culture. The starch amounts accumulated by the two independant wild-type strains are displayed as black squares and diamonds, the ones from the two mutant progeny are displayed as white squares and diamonds. 5 (a) 1 2 1 (b) 2 700 1 600 0.5 500 0 0 6 12 18 24 700 1 D (d) 600 0,5 0.5 500 0 0 6 12 18 ABSORBANCE (OPTICAL DENSITY) MAXIMAL ABSORBANCE WAVELENGTH (nm) C (c) 24 ELUTION VOLUME (mL) 6 Supplemental Fig 5.: Functional complementation of the sta4 mutants. The original mutant strains I73 (sta4-1) and I97 (sta4-2) were transformed with the complete PHOB genomic fragment amplified by PCR and cloned in the pSL18 plasmid (see methods). The paromomycin resistant clones were screened by zymogram (a). Three complementated strains were obtained from 23 resistant clones for I97 and 4 complementated strains from 15 resistant clones for I73. The gel in (a) show the presence of PhoB in the complementated strain I97C1 (lane 2) and the lack of this enzyme activity in a resistant clone that were not successfully complementated (lane 1). RNA from these two strains were extracted and used to amplify the specific 736 bp PHOB cDNA fragment used for cosegregation study (b). This product was visualisable only in the complementated strain I97C1 (lane 2). (c) and (d) correspond respectively to the CL-2B chromatograms obtained with starch extracted with the two previously analysed strains. Starch accumulated by the resistant clone that was not complementated (C) display the typical high-amylose phenotype observed in the original I97 mutant strain (Fig. 3b). Starch extracted from the I97C1 complementated strain (d) is identical to the one produced by a wild-type reference (see Fig. 3a). 7 (a) Starch (µg/106 cells) 70 35 0 0 (b) 6 12 18 24 30 Time (hours) Tub PhoB Supplemental Fig 6: Diurnal behaviour of PHOB mRNA. Chlamydomonas reinhardtii wild-type strain 137C was grown under 12h-light/12h-dark cycle with 2 % (v/v) CO2 constant bubbling during 4 days. Cells were then collected every three hours and crude extracts were prepared from each sample. Half of each samples was used to extract starch. The starch content (in µg per million cells) is displayed in (a). Total RNA were extracted from the other half of the samples. One microgam of total RNA were used to amplify both a fragment of the beta tubulin gene of Chlamydomonas and a specific fragment of PHOB cDNA (see methods). The PCR products obtained were then analyzed on a 1 % agarose gel containing 8 ethidium bromide (b). The black and white bars at the top of the figure represents respectively the dark and the light period. 9