Affinity purification of diverse plant and human 14-3-3-binding partners.

Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants. Our results also suggest that the current perception that 14-3-3s bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen because of the intensity of research on mammalian signalling and for technical reasons.

14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells.

Despite 14-3-3 proteins being implicated in the control of the eukaryotic cell cycle, metabolism, cell signalling and survival, little is known about the global regulation or functions of the phosphorylation-dependent binding of 14-3-3s to diverse target proteins. We identified Arabidopsis cytosolic proteins that bound 14-3-3s in competition with a 14-3-3-binding phosphopeptide, including nitrate reductase, glyceraldehyde- 3-phosphate dehydrogenase, a calcium-dependent protein kinase, sucrose-phosphate synthase (SPS) and glutamyl-tRNA synthetase. Remarkably, in cells starved of sugars or fed with non-metabolizable glucose analogues, all 14-3-3 binding was lost and the target proteins were selectively cleaved into proteolytic fragments. 14-3-3 binding reappeared after several hours of re-feeding with sugars. Starvation-induced degradation was blocked by 5-amino imidazole-4-carboxamide riboside (which is converted to an AMP-mimetic) or the protease inhibitor MG132 (Cbz-leu-leu-leucinal). Extracts of sugar-starved (but not sugar-fed) Arabidopsis cells contained an ATP-independent, MG132-sensitive, neutral protease that cleaved Arabidopsis SPS, and the mammalian 14-3-3-regulated transcription factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was blocked by binding to 14-3-3s. The finding that 14-3-3s participate in a nutrient-sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.

Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major.

A cell-free system based on washed Leishmania major membranes was labelled with GDP-[3H]Man in the presence of synthetic glucosaminyl-phosphatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). In both cases, the major radiolabelled products were Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1, 2-dipalmitoylglycerol) and Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1-palmitoyl-2-lyso-glycerol), to which an additional d-mannose residue was added when a chase with an excess of GDP-Man was performed. The L. major cell-free system can therefore be used to observe the actions of four enzymes, namely GlcNAc-PI de-N-acetylase, Dol-P-Man-GlcN-PI alpha 1-4-mannosyltransferase, a phospholipase A2-like activity and a second alpha-mannosyltransferase activity. The substrate specificities of the first two of these enzymes were studied using a series of substrate analogues. GlcNAc-PI de-N-acetylase was tested against a variety of N-acylated GlcN-PI substrates and was able to cleave N-acetyl and N-propyl groups but not larger groups such as N-butyl, N-isobutyl, N-pentyl and N-hexyl. The Dol-P-Man-GlcN-PI alpha1-4-mannosyltransferase activity required the amino group of the glucosamine residue and the d-configuration of the myo-inositol residue of the GlcN-PI acceptor substrate.