Daily Changes in the Phosphoproteome of the Dinoflagellate Lingulodinium

Bolin Liu

IRBV, University of Montreal, Canada Present address: China National Seed Group Co., Ltd.

Dinoflagellate Lingulodinium polyedra A unicellular protist whose biochemistry and physiology is modulated over the daily cycle by the action of an endogenous circadian clock. This organism serves as a good model system in which to understand the biochemical basis of the observed rythms

-Lingulodinium polyedra is also called Gonyaulax polyedra. An algal bloom of dinoflagellates can result in red tide

Life history of dinoflagellate

Red tide caused by dinoflagellates

Light Dark cycle 0-12 (LD0-12); Light Dark cycle 12-24 (LD12-24)

Day Night

Observed rythms

Two examples in Lingulodinium polyedra where changes in protein amount correlate with the observed rhythm

• Bioluminescence rhythm, where nightly light production is correlated with the amount of two key proteins: luciferase and luciferin binding protein. Both proteins are synthesized at the start of night phase

• Daily rhythm in nitrate reduction which correlates with the amount of a Nitrate Reductase

30%

70%

Phosphoralated proteins

proteins

In plant, arabidopsis has about 1100 kinases and 150 phosphatases. And about 30% of total

proteins are phosphoprylated. They are correlated with biochemical processes such as:

DNA repairation, signal transduction, and transcriptional regulation

0 200 400 600 800 1000 1200

Phosphotase

Kinase

Protein phosphorylation in arabidopsis

Dephosphoprotein Phosphoprotein

Kinase

Phosphotransphorase

By adding ATP

Phosphatase

By removing phosphate group

Protein phosphorylation and dephosphorylation modification

Phosphorylation is a key reversible modification that regulates protein

function

Methods for phosphoprotein detection

• Proteins autoradiography (hot ATP labelling in vivo)

• Immunodetection using antibodies

• Staining gel with a phosphoprotein specific fluorophore (Pro-Q Diamond dye)

• Affinity chromatography (Titanium dioxide)

Methods used in this study

• 1st, Two Dimentional Gel Electrophoresis protocol

2D gel following ProQ Diamond, Coomassie Blue and MS identification

• 2nd, Tryptic Phosphopeptide enrichment protocol

Followed by MS identification

Phosphorylation patterns change over an LD cycle To assess the possibility that Lingulodinium proteins might be differentially phosphorylated at different times under the daily light dark cycle. Samples were taken every four hours, and were resolved by SDS-PAGE

Protein phosphorylation patterns change over an LD cycle.

Protein samples (~50 ug) were prepared for SDS-PAGE from cells isolated at the indicated times (in hours) in a light-dark cycle. The protein samples were resolved by electrophoresis before visualization using ProQ Diamond then Coomassie Blue stains.

A representative gel is shown. Apparent molecular weights are shown at left

Phosphorylation patterns change in 2D gel Protein samples were prepared for 2D-GE from cells taken either at midday (LD6; left hand panels) or midnight (LD18; right hand panels), electrophoresed, and stained sequentially with ProQ Diamond (upper panels) then Coomassie Blue (lower panels). Seven proteins (1-7) that showed at least a 2-fold change in ProQ Diamond staining in three independent experiments and that could be unambiguously assigned to a particular protein after Coomassie Blue staining were excised for protein microsequencing. An additional three highly phosphorylated proteins that did not vary between day and night (a-c) as well as two proteins acting as technical controls (Rub and PCP) were also excised

Identification of Rad24 protein -Shared identity with a Rad24 protein from the dinoflagellate Alexandrium fundyense (in a tBLASTx search of the NCBInr database)

-Suppoted by a good agreement between predicted and measured protein size and isoelectric point

-Rad24 protein is 2.4 times higher in midnight (LD18) than in midday (LD6)

-Rad24 functions in the DNA damage checkpoint pathway

LD18/LD24 kD pI

Linguldinium 2.4 28 4.8

Alexandrium - 27 4.9

Identification of HLCP Shared identity with a chloroplast light harvesting complex protein (LHCP) from the dinoflagellate Heterocapsa triquetra (in a tBLASTx search of the NCBInr database)

-The predicted molecular weight of the Heterocapsa LHCP is higher than that observed on our gels because the predicted protein contains a plastid-directed signal peptide

-The amount of phophorylation of LHCP is 4.3 times higher in midnight (LD18) than in midday (LD6)

-HLCP functions in photosystem I and II

LD18/LD24 kD pI

Linguldinium 4.3 23 4.5

Heterocapsa - 28 7.7

Methods used in this study

• 1st, Two Dimentional Gel Electrophoresis protocol

2D gel following ProQ Diamond, Coomassie Blue and MS identification

• 2nd, Tryptic Phosphopeptide enrichment protocol

Followed by MS identification

Protein samples Samples taken at midday (LD6) and midnight (LD18) were prepared for titanium dioxide (TiO2) chromatography analysis

Results -422 peptides were identified from LC-MS/MS

-54 phosphopeptides were analyzed

-45 different phosphoproteins were identified

Types and motifs of phosphorylation Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine (S) or threonine (T) is the most common

The amino acid context surrounding all phosphorylated serine or threonine residues was used to group the phosphosites into 4 Groups. They are:

Group 1, uncharged/hydrophobic

Group 2, acidic;

Group 3, basic;

Group 4 , S/T followed by proline

Discussion

• 1st, Rad24, identified phosphoprotein from this study, phosphorylated more highly at night is of greatest interest. It is thought to be involved in DNA repair as well as in the DNA damage checkpoint. Phosphorylation of Rad24 is consistent with the observation that S-phase onset occurs at midnight (LD18)

• 2nd, LHCP (light harvesting complex protein) acts to feed energy from light into both photosystem (PS) I and PSII. The phosphorylation of LHCP results in a change in the affinity of the protein for PSI and PSII. The phosphorylated form of LHCP associates preferentially with PSI while the dephosphorylated form has greater affinity for binding to PSII. Here phosphorylation serves as a mechanism to balance the amount of light entering each of the photosystems ensuring the same rate of electron flow through both. In arabidopsis, phosphorylated LHCP dissociate from PSII and associate with PSI during the later part of the night period and decreased throughout the day

Discussion (ctn.)

• 3rd, The phosphopeptide enrichment approach identified three proteins that contain RNA binding domains. That means synthesis of a number of different proteins is controlled translationally

• 4th, A polyketide synthase with at least a 13-fold day/night difference in the amount of phosphopeptide. Polyketides are a diverse family of secondary metabolites and constitute the majority of the dinoflagellate toxins. Toxin production was found to be restricted to the light phase, and the post-translational regulation of polyketide synthase activity may be responsible

• 5th, Two approaches did not identify any of the same phosphoproteins. This presumably reflects the small number of proteins identified using the 2DGE approach

Conclusion

This study provides a proof in principle that an expanded analysis of the phosphoproteome can contribute insight into the daily biochemical changes in Lingulodinium by means of two approaches following MALDI-TOF Mass Spectrometry. Changes in protein phosphorylation may underlie some of rhythmic behavior of Lingludinium.

Acknowledgements

• Dr. David Morse, Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Canada

• Dr. Samuel Chun-Lap Lo, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong SAR, China

• Dr. Daniel P. Matton, IRBV, Université de Montréal

• Dr. B. Franz Lang, Centre Robert Cedergren, Départment de Biochimie,

• Mr. Eric Bonneil at the Institut de Recherche en Immunologie et en Cancérologie (IRIC) for the MS/MS analysis of the TiO2 purified phosphopeptides

• National Science and Engineering Research Council (NSERC) of Canada to DM and BFL (Grant numbers 171382-03 and 194560)

• Reference: Bolin Liu, Samule Lo, et al., Protist, Vol. 163, 746–754, September 2012, Published online date 14 December 2011

Thanks