Download Chlamydomonas reinhardtii strains carrying the stb1-1

(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).
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(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
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ethidium bromide (b). The black and white bars at the top of the figure represents respectively the
dark and the light period.
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