The expanding superfamily of phospholipase A(2) enzymes: classification and characterization.

Phospholipids are emerging as novel second messengers in plant cells. They are rapidly formed in response to a variety of stimuli via the activation of lipid kinases or phospholipases. These lipid signals can activate enzymes or recruit proteins to membranes via distinct lipid-binding domains, where the local increase in concentration promotes interactions and downstream signaling. Here, the latest developments in phospholipid-based signaling are discussed, including the lipid kinases and phospholipases that are activated, the signals they produce, the domains that bind them, the downstream targets that contain them and the processes they control.

Phospholipid-based signaling in plants

Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.

Lipoxygenases - Structure and reaction mechanism.

Molecular enzymology of lipoxygenases.

A phospholipase A2 is transiently synthesized during seed germination and localized to lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.

The N‐terminal β‐barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies

T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP-1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species.

Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls.

The invention relates to an isolated nucleic acid sequence which codes for a polypeptide and consists of a combination of the nucleic acid sequences of a biosynthesis nucleic acid sequence of the fatty acid or lipid metabolism and the following nucleic acids: a) a nucleic acid sequence having the sequence as represented in SEQ ID NO: 1, b) nucleic acid sequences which derive from the degenerated genetic code of the nucleic acid sequence as represented in SEQ ID NO: 1, c) derivatives of the nucleic acid sequence as represented in SEQ ID NO: 1, said derivatives coding for polypeptides with the amino acid sequence as represented in SEQ ID NO: 2 and being provided with at least 60 % homology in the amino acid level, d) a nucleic acid sequence with the sequence as represented in SEQ ID NO: 3 or the aminoterminal component of the coding area pertaining to said sequence.

The beta-barrel of the lipid body lipoxygenase

Proteasomes are self-compartmentalizing protease complexes [1], which are responsible for the degradation of aged or denatured cellular proteins [2]. In higher organism, they in addition generate immuno-competent peptides from foreign antigens [3].



We are interested in the functional mechanism of the 20S core complex from Thermoplasma acidophilum on the single-molecule level. The proteolytic complex of this organism consists of 28 subunits, which are arranged in four heptameric rings and form a cylindric shaped complex of 15 nm in length and 11 nm in diameter. Due to engineered Histidine tags introduced at defined positions [4], the proteasome complex was reversible and functional pre-oriented on self-assembled lipid chelator surfaces via Ni/NTA [5]. In continuation of this work and to reveal the functional mechanism we now investigate degradation of smaller fluorescent-labelled peptides by immobilized proteasomes on the single molecule level. First results of fluorescence measurements in solution with ensembles of proteasomes show that the artificial peptide is an accepted substrate for degradation even under sub-optimal conditions at room temperature (Fig. 1.).



Single immobilized proteasomes on quartz-supported ultra-flat membranes consistent of Ni/NTA-DMPC-chelator-lipid layers could be visualized by total internal reflection microscopy (TIRF) after excitation of the labelled proteasome by an evanescent field. Fig. 2. shows immobilized αC-His-tagged proteasome, 2.6 fold labelled with Oregon Green 488. We are currently monitoring the co-localization of immobilized proteasomes and substrate by FRET studies and plan to image substrate degradation on the single molecule level.

Christian May

Papers

A phospholipase A2 is transiently synthesized during seed germination and localized to lipid bodies.

The N‐terminal β‐barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies

Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls.

The beta-barrel of the lipid body lipoxygenase

Functional Analysis of Individual Proteasome Complexes on Self-organized Chelator-lipids