Regulated import and degradation of a cytosolic protein in the yeast vacuole.

The key regulatory enzyme in gluconeogenesis, fructose 1,6-bisphosphatase (FBPase) is subject to glucose-stimulated proteolytic degradation in Saccharomyces cerevisiae. This process involves the regulated transfer of FBPase directly from the cytosol into the vacuole or a vacuole-related organelle. Glucose may regulate the production of an FBPase receptor or import factor that is transported to the vacuole through the secretory pathway.     

Engineering galactose-binding activity into a C-type mannose-binding protein.

Calcium-dependent or C-type carbohydrate-recognition domains are homologous protein modules found in a variety of animal lectins. Selective binding of sugars by these domains is essential for glycoprotein clearance, cell-cell adhesion and pathogen neutralization. Although various C-type carbohydrate-recognition domains share sequence identity ranging from 20 to 55%, their sugar-binding characteristics vary widely. The structure of a mannose-binding carbohydrate-recognition domain in complex with a saccharide ligand suggests that two glutamic acid-asparagine pairs are essential determinants of ligand binding by this domain. In C-type lectins that bind galactose with higher affinity than mannose, one of these pairs is replaced by glutamine-aspartic acid. Here we shift the sequence of the mannose-binding protein to correspond to that found in galactose-binding domains in order to test the importance of these residues in sugar-binding selectivity. This simple switch in the position of a single amide group alters the binding activity of the domain so that galactose becomes the preferred ligand.     

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.     

SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer.

Non-clathrin coated vesicles have been implicated in early steps of intercompartmental transport. A distinct set of coat proteins are peripherally associated with the exterior of purified mammalian intra-Golgi transport vesicles. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (beta-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat. Here we describe the characterization of SEC21, an essential gene required for protein transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat protein plays an essential role in intercompartmental transport.     

酵母中青蒿素作用靶点的探索

为了验证前人提出的青蒿素在疟原虫中靶点蛋白的可信性并寻找其直接作用的靶点,用酵母作为模式生物,对候选靶点在酵母中的同源蛋白进行基因改造,研究其对青蒿素抑制作用的影响,并利用与琼脂糖偶联的二氢青蒿素(DHA)寻找与之特异性紧密结合的酵母蛋白.研究发现酵母中的同源蛋白似乎没有在青蒿素抑制中起关健作用; 并且未找到能与DHA特异性紧密结合的酵母蛋白.结果表明: 过去提出的青蒿素作用靶点可能不正确,并暗示青蒿素可能没有特异专一的蛋白靶点或同时作用于多个靶点.     

A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility.

Actin is a cytoskeletal protein which is highly conserved across eukaryotic phyla. Actin filaments, in association with a family of myosin motor proteins, are required for cellular motile processes as diverse as vesicle transport, cell locomotion and cytokinesis. Many organisms have several closely related actin isoforms. In addition to conventional actins, yeasts contain actin-related proteins that are essential for viability. We show here that vertebrates also contain an actin-related protein (actin-RPV). Actin-RPV is a major component of the dynactin complex, an activator of dynein-driven vesicle movement, indicating that unlike conventional actins which work in conjunction with myosin motors, actin-RPV may be involved in cytoplasmic movements via a microtubule-based system.     

Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I

In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. We have now cloned and sequenced the gene encoding ABP1p. ABP1p is a novel protein with a 50 amino-acid C-terminal domain that is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. We also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton.