In silico modeling of the Moniliophthora perniciosa Atg8 protein.

Autophagy is defined as an intracellular system of lysosomal degradation in eukaryotic cells, and the genes involved in this process are conserved from yeast to humans. Among these genes, ATG8 encodes a ubiquitin-like protein that is conjugated to a phosphatidylethanolamine (PE) membrane by the ubiquitination system. The Atg8p-PE complex is important in initiating the formation of the autophagosome and thus plays a critical role in autophagy. In silico modeling of Atg8p of Moniliophthora perniciosa revealed its three-dimensional structure and enabled comparison with its Saccharomyces cerevisiae homologue ScAtg8p. Some common and distinct features were observed between these two proteins, including the conservation of residues required to allow the interaction of α-helix1 with the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implying particular roles in selecting specific binding partners. The proposed structure was validated by the programs PROCHECK 3.4, ANOLEA, and QMEAN, which demonstrated 100% of amino acids located in favorable regions with low total energy. Our results showed that MpAtg8p contains the same functional domains (3 α-helices and 4 ß-sheets) and is similar in structure as the ScAtg8p yeast. Both proteins have many conserved sequences in common, and therefore, their proposed three-dimensional models show similar configuration.

Purification and characterization of the chaperone-like Hsp26 from Saccharomyces cerevisiae.

sHsps are ubiquitous ATP-independent molecular chaperones, which efficiently prevent the unspecific aggregation of non-native proteins. Here, we described the purification of the small heat shock protein Hsp26 from a Saccharomyces cerevisiae strain harboring a multicopy plasmid carrying HSP26 gene under the control of its native promoter. A 26 kDa protein was purified to apparent homogeneity with a recovery of 74% by a very reproducible three steps procedure consisting of ethanol precipitation, sucrose gradient ultracentrifugation, and heat inactivation of residual contaminants. The purified polypeptide was unequivocally identified as Hsp26 using a specific Hsp26 polyclonal antibody as a probe. The analysis of the purified protein by electron microscopy revealed near spherical particles with a diameter of 12.0 nm (n=57, standard deviation +/-1.6 nm), displaying a dispersion in size ranging from 9.2 to 16.1 nm, identical to Methanococcus jannaschii Hsp16.5 and in the range of the size estimated for yeast Hsp26, in a previous report. Purified yeast Hsp26 was able to suppress 72% of the heat-induced aggregation of citrate synthase at a ratio of 1:1 (Hsp26 24-mer complex to citrate synthase dimer), and 86% of the heat-induced aggregation of lysozyme at a molar ratio of 1:16 (Hsp26 24-mer complex to lysozyme monomer). In conclusion, the Hsp26 protein purified as described here has structure and activity similar to the previously described preparations. As advantages, this new protocol is very reproducible and requires simple apparatuses which are found in all standard biochemistry laboratories.

Three-dimensional reconstruction of the Saccharomyces cerevisiae multidrug resistance protein Pdr5p.

Pdr5p, the major multidrug exporter in Saccharomyces cerevisiae, is a member of the ATP-binding cassette (ABC) superfamily. Pdr5p shares similar mechanisms of substrate recognition and transport with the human MDR1-Pgp, despite an inverted topology of transmembrane and ATP-binding domains. The hexahistidine-tagged Pdr5p multidrug transporter was highly overexpressed in yeast strains where other ABC genes have been deleted. After solubilization and purification, the 160-kDa recombinant Pdr5p has been reconstituted into a lipid bilayer. Controlled detergent removal from Pdr5p-lipid-detergent micelles allowed the production of peculiar square-shaped particles coexisting with liposomes and proteoliposomes. These particles having 11 nm in side were well suited for single particle analysis by electron microscopy. From such analysis, a computed volume has been determined at 25-A resolution, giving insight into the structural organization of Pdr5p. Comparison with the reported structures of different bacterial ABC transporters was consistent with a dimeric organization of Pdr5p in the square particles. Each monomer was composed of three subregions corresponding to a membrane region of about 50 A in height that joins two well separated protruding stalks of about 40 A in height, ending each one with a cytoplasmic nucleotide-binding domain (NBD) lobe of about 50-60 A in diameter. The three-dimensional reconstruction of Pdr5p revealed a close arrangement and a structural asymmetric organization of the two NBDs that appeared oriented perpendicularly within a monomer. The existence of different angular positions of the NBDs, with respect to the stalks, suggest rotational movements during the catalytic cycle.