Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae

Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p, Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta -lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae. (C) 2001 Academic Press

Wild-type and mutant alpha-synuclein induce a multi-component gene expression profile consistent with shared pathophysiology in different transgenic mouse models of PD

The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons residing in the substantia nigra with devastating consequences for normal movement. One important gene involved in both familial and sporadic PD is \u3b1-synuclein. We have generated three strains of \u3b1-synuclein transgenic mice to study the pathologic consequences of the targeted expression of mutant or wild-type human \u3b1-synuclein in a model system. We have analyzed gene expression patterns in these mice using high throughput microarrays in anatomical regions implicated in disease (substantia nigra and brainstem). Our study reveals gene dosage-dependent dysregulation of several genes important for the dopaminergic phenotype in mice over-expressing wild-type human \u3b1-synuclein in the substantia nigra at time points preceding neuronal cell death. Analysis of mutant \u3b1-synuclein mice at a time point when pathology is advanced reveals several new candidate genes that may play a role in neuronal demise and/or protein accumulation

Identification in silico of putative damage responsive elements (DRE) in promoter regions of the yeast genome

We report an in silico analysis to identify nucleotide sequence motifs in DNA repair genes that may define a binding site for regulatory proteins during the induction of those genes by mutagens. The damage responsive elements (DRE) weight matrix generated in this analysis was used to search for homologous sequences in the promoter region of all genes, including putative gene and hypothetical open reading frames (ORFs), in the Saccharomyces Genome Data Base (SGD). The results demonstrated that over one third of the yeast genes in the database presented at least one 15-bp sequence in their promoter region with 85% or more of similarity to the DRE consensus sequence. The presence of the DRE sequence in the promoter region of regulatory genes and its high similarity to other well reported DNA binding sites points to its involvement in the general regulation of not only DNA repair genes but yeast genes in general