An amphitropic cAMP-binding protein in yeast mitochondria

ABSTRACT: We describe the first example of a mitochondrial protein with a covalently attached phos-phatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cereuisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified CAMP-binding protein through a phospholipase activity. Only in recent years specific fatty acids have been recog-nized to play important roles in the association of proteins with membranes. Both noncovalent and covalent interactions be-tween fatty acids and proteins have been reported. Among the latter are GTP-binding proteins (Molenaar et al., 1988)

Analysis of the Effects of Polymorphism on Pollen Profilin Structural Functionality and the Generation of Conformational, T- and B-Cell Epitopes

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases. [EN]This study was supported by the following European Regional Development Fund co-financed grants: MCINN BFU 2004-00601/BFI, BFU 2008-00629, BFU2011-22779, CICE (Junta de Andalucía) P2010-CVI15767, P2010-AGR6274 and P2011-CVI-7487, and by the coordinated project Spain/Germany MEC HA2004-0094. JCJ-L thanks Spanish CSIC and the European Marie Curie research program for his I3P-BPD-CSIC, and PIOF-GA-2011-301550 grants, respectively.Peer reviewe

Characterization of Profilin Polymorphism in Pollen with a Focus on Multifunctionality

Profilin, a multigene family involved in actin dynamics, is a multiple partners-interacting protein, as regard of the presence of at least of three binding domains encompassing actin, phosphoinositide lipids, and poly-L-proline interacting patches. In addition, pollen profilins are important allergens in several species like Olea europaea L. (Ole e 2), Betula pendula (Bet v 2), Phleum pratense (Phl p 12), Zea mays (Zea m 12) and Corylus avellana (Cor a 2). In spite of the biological and clinical importance of these molecules, variability in pollen profilin sequences has been poorly pointed out up until now. In this work, a relatively high number of pollen profilin sequences have been cloned, with the aim of carrying out an extensive characterization of their polymorphism among 24 olive cultivars and the above mentioned plant species. Our results indicate a high level of variability in the sequences analyzed. Quantitative intra-specific/varietal polymorphism was higher in comparison to inter-specific/cultivars comparisons. Multi-optional posttranslational modifications, e.g. phosphorylation sites, physicochemical properties, and partners-interacting functional residues have been shown to be affected by profilin polymorphism. As a result of this variability, profilins yielded a clear taxonomic separation between the five plant species. Profilin family multifunctionality might be inferred by natural variation through profilin isovariants generated among olive germplasm, as a result of polymorphism. The high variability might result in both differential profilin properties and differences in the regulation of the interaction with natural partners, affecting the mechanisms underlying the transmission of signals throughout signaling pathways in response to different stress environments. Moreover, elucidating the effect of profilin polymorphism in adaptive responses like actin dynamics, and cellular behavior, represents an exciting research goal for the future

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca 2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP γ S in permeabilized cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44280/1/10571_2004_Article_BF00713279.pd