Rapid Plasma Membrane Anchoring of Newly Synthesized p59 fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59^fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [³⁵S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH₂-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gα_o-, Gα_s-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.     

The response of tomato roots (Lycopersicon esculentum Mill.) to iron deficiency stress: alterations in the pattern of protein synthesis

Dicotyledon plants adapt to iron (Fe) deficiency through a seriesof reactions that increase the ability of the plant to assimilateFe and to increase the efficiency of Fe utilization. In an attemptto gain an insight into these adaptive processes, the specificchanges in protein synthesis associated with the onset of theFe deficiency response in tomato roots (Lycopersicon esculentumMill cv. Rutgers) have been investigated. Roots were grown underFe—sufficient and —deficient conditions, and thepattern of protein synthesis was analysed by in vitro translationof root mRNA and by in vivo labelling of root proteins. Polypeptideswere resolved by two—dimensional polyacrylamide gel elec—trophoresis.Seven polypeptides were identified by in vitro translation,whose synthesis was significantly increased during Fe deficiency.The increase was probably specific to Fe deficiency in thatthe polypep—tide synthesis was not increased during phosphatedeficiency stress, was less prominent following prolonged Fedeficiency and was decreased following re—supply of Feto the hydroponic medium. The pattern of in vitro translation of mRNA isolated from Fe—deficientroots was compared to the results obtainedin vivo followingradiolabelling of proteins. In these analyses, eight polypeptideswere identified, tentatively including the seven polypeptidespreviously identified by in vitro translation. All polypeptideswere characterized with regard to molecular mass and pl andtheir localization in the cell, whether being membrane boundor soluble. It is suggested that members of this group of polypeptidesare involved in the response of the root to Fe deficiency: althoughtheir functions remain to be identified. Key words: In vitro protein synthesis, iron, iron deficiency, root, 2-dimensional PAGE     

Detection of expiratory flow limitation during exercise in COPD patients

Koulouris, Nickolaos G., Ioanna Dimopoulou, PäiviValta, Richard Finkelstein, Manuel G. Cosio, and J. Milic-Emili.Detection of expiratory flow limitation during exercise in COPDpatients. J. Appl. Physiol. 82(3):723-731, 1997.The negative expiratory pressure (NEP) method wasused to detect expiratory flow limitation at rest and at differentexercise levels in 4 normal subjects and 14 patients with chronicobstructive pulmonary disease (COPD). This method does not requireperformance of forced expirations, nor does it require use of bodyplethysmography. It consists in applying negative pressure (5cmH₂O) at the mouth during early expiration and comparing the flow-volume curve of the ensuing expiration with that of the preceding control breath. Subjects in whomapplication of NEP does not elicit an increase in flow during part orall of the tidal expiration are considered flow limited. The fournormal subjects were not flow limited up to 90% of maximal exercisepower output(_max).Five COPD patients were flow limited at rest, 9 were flow limited atone-third _max, and 12 were flow limited at two-thirds_max. Whereasin all patients who were flow limited at rest the maximalO₂ uptake was below the normallimits, this was not the case in most of the other patients. Inconclusion, NEP provides a rapid and reliable method to detectexpiratory flow limitation at rest and during exercise.

     

Clinical and Molecular Characterization of Patients with Distal 11q Deletions

Jacobsen syndrome is caused by segmental aneusomy for the distal end of the long arm of chromosome 11. Typical features include mild to moderate psychomotor retardation, trigonocephaly, facial dysmorphism, cardiac defects, and thrombocytopenia, though none of these features are invariably present. To define the critical regions responsible for these abnormalities, we studied 17 individuals with de novo terminal deletions of 11q. The patients were characterized in a loss-of-heterozygosity analysis using polymorphic dinucleotide repeats. The breakpoints in the complete two-generation families were localized with an average resolution of 3.9 cM. Eight patients with the largest deletions extending from 11q23.3 to 11qter have breakpoints, between D11S924 and D11S1341. This cytogenetic region accounts for the majority of 11q⁻ patients and may be related to the FRA11B fragile site in 11q23.3. One patient with a small terminal deletion distal to D11S1351 had facial dysmorphism, cardiac defects, and thrombocytopenia, suggesting that the genes responsible for these features may lie distal to D11S1351. Twelve of 15 patients with deletion breakpoints as far distal as D11S1345 had trigonocephaly, while patients with deletions distal to D11S912 did not, suggesting that, if hemizygosity for a single gene is responsible for this dysmorphic feature, the gene may lie distal to D11S1345 and proximal to D11S912.     

Ribonuclease P from plant nuclei and photosynthetic organelles

RNase P consists of both protein and RNA subunits in all organisms and organelles investigated so far, with the exception of chloroplasts and plant nuclei where no enzyme-associated RNA has been detected to date. Studies on substrate specificity revealed that cleavage by plant nuclear RNase P is critically dependent on a complete and intact structure of the substrate. No clearcut answer is yet possible regarding the order of processing events at the 5 or 3 end of tRNAs in the case of nuclear or chloroplast processing enzymes. RNase P from a phylogenetically ancient photosynthetic organelle will be discussed in greater detail: The enzyme from theCyanophora paradoxa cyanelle is the first RNase P from a photosynthetic organelle which has been shown to contain an essential RNA subunit. This RNA is strikingly similar to its counterpart from cyanobacteria, yet it lacks catalytic activity. Properties of the holoenzyme suggest an intermediate position in RNA enzyme evolution, with an eukaryotic-type, inactive RNA and a prokaryotic-type small protein subunit. The possible presence of an RNA component in RNase P from plant nuclei and modern chloroplasts will be discussed, including a critical evaluation of some criteria that have been frequently applied to elucidate the subunit composition of RNase P from different organisms.Abbreviations RNase P Ribonuclease P - (pre-)tRNA transfer ribonucleic acid (precursor) - tRNA ^Ser (- ^Tyr , - ^Phe ) transfer ribonucleic acid specific for serine (tyrosine, phenylalanine) - CyRP RNA RNA component of cyanelle RNase P     

Resolution of sensory and mucoid glycoconjugates with terminal α-galactose residues in the mucomicrovillar complex of the vomeronasal sensory epithelium by dual confocal laser scanning microscopy

The organization of the mucomicrovillar complex of the vomeronasal sensory epithelium of adult rats was examined using confocal laser scanning microscopy. In specimens labeled with the FITC-conjugated isolectin B₄ of Bandeiraea simplicifolia, which recognizes terminal -galactose sugar residues of glycoconjugates, we demonstrated that the mucomicrovillar complex was composed of islet-like structures with a high-density -galactose core. The mucomicrovillar complex was further resolved into sensory and mucoid components in double-labeling and dual scanning experiments. The sensory component, which consists of the dendritic terminals of olfactory marker protein-immunoreactive vomeronasal receptor neurons, contained cytosolic glycoconjugates with terminal -galactose sugar residues. The extracellular mucoid component consisted of glycoconjugates containing terminal -galactose derived from the glands associated with the vomeronasal organ. These results demonstrated the complex microchemical organization of the sensory and mucoid components of the mucomicrovillar complex.     

Plasmid rolling circle replication and its control

Abstract This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.     

Mapping of the spectral density function of a Cα−Hα bond vector from NMR relaxation rates of a 13C-labelled α-carbon in motilin

Summary The peptide hormone motilin was synthesised with a ¹³C-enriched -carbon in the leucine at position 10. In aqueous solution, six different relaxation rates were measured for the ¹³C–H fragment as a function of temperature and with and without the addition of 30% (v/v) of the cosolvent d ₂-1,1,1,3,3,3-hexafluoro-2-propanol (HFP). The relaxation rates were analysed employing the spectral density mapping technique introduced by Peng and Wagner [(1992) J. Magn. Reson., 98, 308–322] and using the model-free approach by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570]. The fit to various models of dynamics was also considered. Different procedures to evaluate the overall rotational correlation time were compared. A single exponential time correlation function was found to give a good fit to the measured spectral densities only for motilin in 30% (v/v) HFP at low temperatures, whereas at high temperatures in this solvent, and in D₂O at all temperatures, none of the considered models gave an acceptable fit. A new empirical spectral density function was tested and found to accurately fit the experimental spectral density mapping points. The application of spectral density mapping based on NMR relaxation data for specific ¹³C–¹H vector is shown to be a highly useful method to study biomolecular dynamics. Advantages are high sensitivity, high precision and uniform sampling of the spectral density function over the frequency range.Abbreviations CD circular dichroism - NOE nuclear Overhauser enhancement - NOESY two-dimensional NOE spectroscopy - INEPT insensitive nuclei enhanced by polarisation transfer - DANTE delays alternating with nutations for tailored excitation - WALTZ-16 wideband, alternating phase, low-power technique for zero residual splitting - FID Free induction decay - ppm parts per million - TSPA 3-trimethylsilyl-(3,3,2,2-d)-propionic acid - HFP d ₂-1,1,1,3,3,3-hexafluoro-2-propanol - CPMG Carr-Purcell-Meiboom-Gill - TFD time-resolved fluorescence depolarisation - CSA chemical shift anisotropy Partly presented at the symposium Dynamics and Function of Biomolecules, Szeged, Hungary, July 31–August 2, 1993.