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In the paper by Becerra et al., published in Molecular Microbiology volume 43, number 3, pp. 545-555, the rox3 mutant strain used was not a deletion, lethal in the selected aGH1 background, but the rox3-182 allele with a nonsense mutation at codon 129, gal1∆152,.

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.

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Saccharomyces cerevisiae and Kluyveromyces lactis are considered to be the prototypes of two distinct metabolic models of facultatively-aerobic yeasts: Crabtree-positive/fermentative and Crabtree-negative/respiratory, respectively. Our group had previously proposed that one of the molecular keys supporting this difference lies in the mechanisms involved in the reoxidation of the NADPH produced as a consequence of the activity of the pentose phosphate pathway. It has been demonstrated that a significant part of this reoxidation is carried out in K. lactis by mitochondrial external alternative dehydrogenases which use NADPH, the enzymes of S. cerevisiae being NADH-specific. Moreover, the NADPH-dependent pathways of response to oxidative stress appear as a feasible alternative that might co-exist with direct mitochondrial reoxidation.

Genome duplication, after the divergence of Saccharomyces cerevisiae from Kluyveromyces lactis along evolution, has been proposed as a mechanism of yeast evolution from strict aerobics, such as Candida albicans, to facultatives/fermentatives, such as S. cerevisiae. This feature, together with the preponderance of respiration and the use of the pentose phosphate pathway in glucose utilization, makes K. lactis a model yeast for studies related to carbon and oxygen metabolism. In this work, and based on the knowledge of the sequence of the genome of K. lactis, obtained by the Génolevures project, we have constructed DNA arrays from K. lactis including a limited amount of selected probes. They are related to the aerobiosis-hypoxia adaptation and to the oxidative stress response, and have been used to test changes in mRNA levels in response to hypoxia and oxidative stress generated by H(2)O(2). The study was carried out in both wild-type and rag2 mutant K. lactis strains in which glycolysis is blocked at the phosphoglucose isomerase step. This approach is the first analysis carried out in K. lactis for the majority of the genes selected.

We examined several strategies for the secretion of Kluyveromyces lactis beta-galactosidase into the culture medium, in order to facilitate the downstream processing and purification of this intracellular enzyme of great industrial interest. We constructed plasmids by fusing the LAC4 gene or engineered variants to the secretion signal of the K.lactis killer toxin or to the secretion signal of the Saccharomyces cerevisiae alpha-factor. With these plasmids we transformed strains of the yeasts K.lactis and S.cerevisiae, respectively and tested beta-galactosidase extracellular activity in different culture media. We achieved partial secretion of beta-galactosidase in the culture medium since the high molecular weight and oligomeric nature of the enzyme, among other factors, preclude full secretion. The percentage of secretion was improved by directed mutagenesis of the N-terminus of the protein. We developed several deletion mutants which helped us to propose structure-function relationships by comparison with the available data on the homologous Escherichia coli beta-galactosidase. The influence of the culture conditions on heterologous beta-galactosidase secretion was also studied.