Magnus Rosenquist

A pigment-binding protein essential for regulation of photosynthetic light harvesting.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plants capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

Phosphorylation of Thr-948 at the C Terminus of the Plasma Membrane H+-ATPase Creates a Binding Site for the Regulatory 14-3-3 Protein

The plant plasma membrane H+-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H+-ATPase–14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT948V, at the C terminus of the H+-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H+-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H+-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H+-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H+-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H+-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H+-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H+-ATPase in vivo. Indeed, replacing Thr-948 in the plant H+-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H+-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H+-ATPase activity in the plant and thus for plant growth.

Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

Abstract. 14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H+ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

Evolution and isoform specificity of plant 14-3-3 proteins.

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.

Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants.

Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.

Identification of a novel calreticulin isoform (Crt2) in human and mouse

Calreticulin is a Ca(2+)-binding chaperone localized mainly in the endoplasmic/sarcoplasmic reticulum in all higher organisms. To date, only one calreticulin isoform has been identified in human and mouse. Here we report a novel calreticulin isoform (Crt2) in human and mouse, with 53 (human) and 49% (mouse) identity to the previously identified calreticulin in respective species. The gene encoding the novel human calreticulin isoform spans 17 kb of genomic DNA and is expressed in testis, showing a similar expression as the chaperone calmegin. Phylogenetic analysis shows that two or more calreticulin (crt) genes are present both in plants and in mammals. The duplication of the crt gene in human and mouse suggests functional diversity, and variations in expression patterns among calreticulins. Two novel calreticulin (Crt2) isoforms, with high homology to the human and mouse calreticulin isoform (Crt2), were also identified in pig and rat via expressed sequence tags.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alters Mitochondrial Membrane Lipids

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to have selective antitumor activity. TRAIL induces ubiquitous pathways of cell death in which caspase activation is mediated either directly or via the release of apoptogenic factors from mitochondria; however, the precise components of the mitochondrial signaling pathway have not been well defined. Notably, mitochondria constitute an important target in overcoming resistance to TRAIL in many types of tumors. Bid is considered to be fundamental in engaging mitochondria during death receptor-mediated apoptosis, but this action is dependent on mitochondrial lipids. Here, we report that TRAIL signaling induces an alteration in mitochondrial membrane lipids, particularly cardiolipin. This occurs independently of caspase activation and primes mitochondrial membranes to the proapoptotic action of Bid. We unveil a link between TRAIL signaling and alteration of membrane lipid homeostasis that occurs in parallel to apical caspase activation but does not take over the mode of cell death because of the concurrent activation of caspase-8. In particular, TRAIL-induced alteration of mitochondrial lipids follows an imbalance in the cellular homeostasis of phosphatidylcholine, which results in an elevation in diacylglycerol (DAG). Elevated DAG in turn activates the delta isoform of phospholipid-dependent serine/threonine protein kinase C, which then accelerates the cleavage of caspase-8. We also show that preservation of phosphatidylcholine homeostasis by inhibition of lipid-degrading enzymes almost completely impedes the activation of pro-caspase-9 while scarcely changing the activation of caspase-8.