scholarly journals A key role for phosphorylation of PsbH in the biogenesis and repair of photosystem II in Chlamydomonas

2019 ◽  
Author(s):  
Alexis Riché ◽  
Linnka Lefebvre-Legendre ◽  
Michel Goldschmidt-Clermont
Keyword(s):  
Protein Kinase ◽  
Photosystem Ii ◽  
Chloroplast Transformation ◽  
Site Directed Mutagenesis ◽  
Alanine Substitution ◽  
Evolutionarily Conserved ◽  
Delayed Recovery ◽  
Psii Subunits ◽  
A Minor

ABSTRACTPhosphorylation of the core subunits of photosystem II (PSII) is largely governed by a protein kinase and an antagonistic protein phosphatase. In plants the respective mutants show alterations in the architecture of thylakoid membranes and in the repair of PSII after photo-inhibition. However the protein kinase targets several subunits of PSII, as well as other proteins. To specifically investigate the role of phosphorylation of the different PSII subunits, we used site-directed mutagenesis and chloroplast transformation in Chlamydomonas reinhardtii. Major, evolutionarily-conserved sites of phosphorylation in three components of PSII (CP43, D2 and PsbH) were mutated to replace the corresponding serine or threonine residues with alanine. The alanine substitution mutant of D2 had no apparent phenotype, while the mutant of CP43 presented a minor delay in recovery from photo-inhibition. Alanine substitutions of the phosphorylation sites in PsbH had significant effects on the accumulation of PSII or on its recovery from photo-inhibition. When mutations in two of the target subunits were combined through a second cycle of chloroplast transformation, the strongest phenotype was observed in the mutant lacking phosphorylation of both PsbH and CP43, which showed delayed recovery from photo-inhibition. Surprisingly this phenotype was reversed in the mutant defective for phosphorylation of all three subunits. Our analysis indicates a prominent role for the N-terminus of PsbH in the stable accumulation of PSII and of PsbH phosphorylation in its repair cycle.SIGNIFICANCE STATEMENTTo specifically investigate the role of PSII phosphorylation, alanine-substitution mutants of the major phospho-sites in the subunits of PSII were generated individually or in combinations using chloroplast transformation. PSII assembly was defective in some of the PsbH mutants. PSII repair after photo-inhibition was delayed most strongly in the mutant lacking phosphorylation of both PsbC (CP43) and PsbH.

scholarly journals Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

2016 ◽  
Vol 473 (11) ◽  
pp. 1605-1615 ◽  
Author(s):  
Sara R. Costa ◽  
Magdalena Marek ◽  
Kristian B. Axelsen ◽  
Lisa Theorin ◽  
Thomas G. Pomorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Evolutionarily Conserved ◽  
A Site ◽  
P Type ◽  
Cell Division Control

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.

scholarly journals Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria

2017 ◽  
Vol 372 (1730) ◽  
pp. 20160394 ◽  
Author(s):  
Martina Bec̆ková ◽  
Jianfeng Yu ◽  
Vendula Krynická ◽  
Amanda Kozlo ◽  
Shengxi Shao ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Minor Component ◽  
Crop Plants ◽  
Irreversible Damage ◽  
X Ray Crystallography ◽  
Selective Degradation ◽  
Ftsh Protease ◽  
Psii Subunits ◽  
A Minor

One strategy for enhancing photosynthesis in crop plants is to improve their ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid-embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His-tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero-oligomeric complex involved in PSII repair. We show using X-ray crystallography that Psb29 from Thermosynechococcus elongatus has a unique fold consisting of a helical bundle and an extended C-terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production of reactive oxygen species, the loss of chloroplast function and cell death. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers

2001 ◽  
Vol 353 (2) ◽  
pp. 345-355 ◽  
Author(s):  
Richard K. HUGHES ◽  
Stuart I. WEST ◽  
Andrzej R. HORNOSTAJ ◽  
David M. LAWSON ◽  
Shirley A. FAIRHURST ◽  
...  
Keyword(s):  
Potato Tuber ◽  
Critical Role ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Eicosatetraenoic Acid ◽  
Positional Specificity ◽  
Binding Pockets ◽  
Plant Lipoxygenases ◽  
A Minor

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

scholarly journals The regulation of AMP-activated protein kinase by phosphorylation

2000 ◽  
Vol 345 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Silvie C. STEIN ◽  
Angela WOODS ◽  
Neil A. JONES ◽  
Matthew D. DAVISON ◽  
David CARLING
Keyword(s):  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Aspartic Acid Residue ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Mutant Complex

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.

scholarly journals PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

2018 ◽  
Vol 47 (4) ◽  
pp. 1742-1750 ◽  
Author(s):  
Annette Buur Steffensen ◽  
Sofia Hammami Bomholtz ◽  
Martin Nybo Andersen ◽  
Jesper Velgaard Olsen ◽  
Nancy Mutsaers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Protein Kinase D ◽  
Protein Levels ◽  
Whole Cell Patch Clamp

Background/Aims: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation. Methods: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. Results: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1. Conclusions: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Role of PsbV-Tyr137 in photosystem II studied by site-directed mutagenesis in the thermophilic cyanobacterium Thermosynechococcus vulcanus

2020 ◽  
Vol 146 (1-3) ◽  
pp. 41-54 ◽  
Author(s):  
Yanan Xiao ◽  
Qingjun Zhu ◽  
Yanyan Yang ◽  
Wenda Wang ◽  
Tingyun Kuang ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Thermophilic Cyanobacterium

scholarly journals Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

1993 ◽  
Vol 122 (3) ◽  
pp. 601-612 ◽  
Author(s):  
JA Porter ◽  
C Montell
Keyword(s):  
Protein Kinase ◽  
Retinal Degeneration ◽  
Kinase Domain ◽  
Specific Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Multiple Functions

The Drosophila ninaC locus encodes a rhabdomere specific protein (p174) with linked protein kinase and myosin domains, required for a wild-type ERG and to prevent retinal degeneration. To investigate the role for linked kinase and myosin domains, we analyzed mutants generated by site-directed mutagenesis. Mutation of the kinase domain resulted in an ERG phenotype but no retinal degeneration. Deletion of the myosin domain caused a change in the subcellular distribution of p174 and resulted in both ERG and retinal degeneration phenotypes. Temperature-sensitive mutations in the myosin domain resulted in retinal degeneration, but no ERG phenotype. These results indicated that the ERG and retinal degeneration phenotypes were not strictly coupled suggesting that the myosin domain has multiple functions. We propose that the role of the kinase domain is to regulate other rhabdomeric proteins important in phototransduction and that the myosin domain has at least two roles: to traffic the kinase into the rhabdomeres and to maintain the rhabdomeres.

The Effect of Path Length and Display Size on Memory for Spatial Information

2012 ◽  
Vol 59 (3) ◽  
pp. 147-152 ◽  
Author(s):  
Katherine Guérard ◽  
Sébastien Tremblay
Keyword(s):  
Path Length ◽  
Potential Role ◽  
Spatial Information ◽  
Recall Performance ◽  
Minor Role ◽  
Length Effect ◽  
Serial Memory ◽  
Fixed Reference ◽  
A Minor

In serial memory for spatial information, some studies showed that recall performance suffers when the distance between successive locations increases relatively to the size of the display in which they are presented (the path length effect; e.g., Parmentier et al., 2005) but not when distance is increased by enlarging the size of the display (e.g., Smyth & Scholey, 1994). In the present study, we examined the effect of varying the absolute and relative distance between to-be-remembered items on memory for spatial information. We manipulated path length using small (15″) and large (64″) screens within the same design. In two experiments, we showed that distance was disruptive mainly when it is varied relatively to a fixed reference frame, though increasing the size of the display also had a small deleterious effect on recall. The insertion of a retention interval did not influence these effects, suggesting that rehearsal plays a minor role in mediating the effects of distance on serial spatial memory. We discuss the potential role of perceptual organization in light of the pattern of results.

Sign in / Sign up

Export Citation Format

Share Document