Laser ablation interfaced with inductively coupled plasma-mass spectrometry is described as a new method to determine the presence of phosphorylated proteins on electrophoresis gel blots. The method was applied to the phosphoprotein beta-casein with good detection levels being observed at 16 pmole. Attempts at using the technique to detect beta-casein on electrophoresis gels are also described.

In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry. Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.

Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites.Calnexin was originally identified and purified as a constituent of a complex of four co-isolated integral membrane proteins, two of which were phosphorylated in microsomes by ER 1 -associated kinase(s) (1). This phosphorylation was exclusively on serine residues and by controlled protease digestion found to be cytosolically oriented (1, 2). As a consequence of the cDNA cloning of this phosphoprotein, it was predicted and subsequently confirmed to be a type I integral membrane protein with extensive sequence similarity in its luminal domain to the ER luminal resident protein, calreticulin (1, 2). Calnexin and then calreticulin were found to be novel molecular chaperones of the ER. These chaperones act as lectins to couple oligosaccharide modifications to newly synthesized N-linked glycoproteins with productive glycoprotein folding. The lectin specificity of these chaperones has been identified as the recognition of high mannose oligosaccharides terminating in monoglucosyl residues linked ␣1-3 (3-13

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

A functionally active analogue of neurotensin, neurotensin(8^13), has been observed whilst bound to the agonistbinding site of the rat neurotensin receptor by nuclear magnetic resonance (NMR). Through the application of slow magic angle sample spinning and high-power proton decoupling, sufficient resolution and sensitivity were obtained in the carbon-13 spectrum to allow an assignment of many of the side chain resonances arising from uniformly carbon-13/nitrogen-15-labelled neurotensin(8^13) whilst bound to the neurotensin receptor. Significant perturbations in carbon-13 chemical shift were observed upon the binding of the neurotensin(8^13) to the receptor. Most importantly significant shifts were observed in both the carboxy terminus and tyrosine side chain of the neurotensin(8^13), suggesting that these sites are important in the interaction of the neurotensin with the agonist-binding site on the neurotensin receptor. Conversely, no perturbations were observed for the carbon-13 sites within the guanidinium groups of the arginine side chains, indicating little interaction with the receptor-binding site, or a shielding of the local environment by the surrounding nitrogen atoms. These NMR observations lend further support to previous structure^activity studies, site-directed mutagenesis and modelling studies of the agonist-binding site of the neurotensin receptor, from which the same specific residues for which NMR perturbations were observed are important for neurotensin receptor activation by neurotensin. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.