Showing papers by "Chris A. Kaiser published in 2002"

Formation and transfer of disulphide bonds in living cells.

Abstract: Protein disulphide bonds are formed in the endoplasmic reticulum of eukaryotic cells and the periplasmic space of prokaryotic cells The main pathways that catalyse the formation of protein disulphide bonds in prokaryotes and eukaryotes are remarkably similar, and they share several mechanistic features The recent identification of new redox-active proteins in humans and yeast that mechanistically parallel the more established redox-active enzymes indicates that there might be further uncharacterized redox pathways throughout the cell

A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p.

Abstract: Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum. The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases. Two pairs of cysteine residues are required for Erv2p activity. The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD. The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.

Identification of Sec36p, Sec37p, and Sec38p: Components of Yeast Complex That Contains Sec34p and Sec35p

Abstract: The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.

Amino acids regulate the intracellular trafficking of the general amino acid permease of Saccharomyces cerevisiae

Abstract: The delivery to the plasma membrane of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by the quality of the nitrogen source in the growth medium. In an effort to define how different nitrogen sources control Gap1p sorting, we find that mutations in GDH1 and GLN1 that decrease the flux through the glutamate and glutamine synthesis pathways result in increased Gap1p sorting to the plasma membrane. Conversely, deletion of MKS1, which increases glutamate and glutamine synthesis, decreases Gap1p sorting to the plasma membrane. Glutamate and glutamine are not unusual in their ability to regulate Gap1p sorting, because the addition of all natural amino acids and many amino acid analogs to the growth medium results in increased Gap1p sorting to the vacuole. Importantly, amino acids have the capacity to signal Gap1p sorting to the vacuole regardless of whether they can be used as a source of nitrogen. Finally, we show that rapamycin does not affect Gap1p sorting, indicating that Gap1p sorting is not directly influenced by the TOR pathway. Together, these data show that amino acids are a signal for sorting Gap1p to the vacuole and imply that the nitrogen-regulated Gap1p sorting machinery responds to amino acid-like compounds rather than to the overall nutritional status associated with growth on a particular nitrogen source.

Subcellular fractionation of secretory organelles.

Abstract: Publisher Summary This chapter discusses the subcellular fractionation of secretory organelles along with a set of cell fractionation protocols used to explore whether a protein might be associated with a membrane compartment and to localize those proteins that are associated with organelles. These procedures complement microscopic localization studies and are usually carried out in parallel. The first step in any localization study is to develop immunological reagents specific for the detection of the protein of interest. This is usually done either by raising antibodies to the recombinant yeast protein expressed in Escherichia coli or by adding an epitope tag to the yeast gene of interest and verifying that the tagged gene is functional. It is also important to work out methods for the detection of the protein of interest by immunoblotting. These preliminaries include finding conditions to stabilize the protein of interest against proteolysis in cell extracts to solubilize the protein in a gel-loading buffer and to optimize conditions for electrophoretic transfer to a nitrocellulose membrane for immunoblotting. The chapter extends the general usefulness of the fractionation procedures using reagents commercially available for the detection of markers.