Chris Kaiser - US grants

A fundamental aspect of eukaryotic cell assembly and growth is the transport of newly synthesized proteins from the cytoplasm to one of a number of chemically distinct subcellular compartments. The secretory pathway, a prominent route of intracellular protein transport, delivers both soluble and membrane-bound proteins to the cell surface. Each step in this transport process is mediated by cargo-carrying vesicles that bud from the membrane of one compartment and fuse with the membrane of the next. The yeast Saccharomyces cerevisiae has a secretory pathway much like that of mammalian cells. An important class of genes (SEC) that drive the process of vesicular protein transport have been identified through the use of mutants that disrupt the secretory process. Little is known of the precise function of most of these genes. Dr. Kaiser proposes to develop a new method to study the function of SEC gene products by determining their precise intracellular location. The yeast Pichia pastoris is similar to S. cerevisiae in many respects except that P. pastoris cells contain prominent and morphologically well defined Golgi complexes. Methods will be devised for the isolation of P. pastoris homologs of SEC genes and the localization of their products in P. pastoris cells by antibody staining and electron microscopy. In addition, Dr. Kaiser will develop genetic screens to identify new SEC genes. The products of these new genes will also be localized in P. pastoris. Finally, by using dominant negative mutations in SEC genes, a general method for expressing secretory defects in different cell types will be tested. This method will be particularly useful for studying the function of secretory genes in diploid organisms. Elucidation of the molecular mechanism of protein transport in yeast will directly contribute to our understanding of the equivalent processes in human cells and may, therefore, speed the development of therapies for diseases caused by those viruses that use the secretory pathway to exit infected cells. In addition, some of the genes identified in this work may be particular to fungal secretory pathway and could provide selective targets for therapy against fungal pathogens.

An important aspect of cell regulation is the controlled delivery of transport proteins to the plasma membrane, allowing the cells capacity to take up small molecules to respond to extracellular signals. Dr. Kaiser's research group proposes a combination of genetic and biochemical experiments in the yeast S. cerevisiae to give fundamental insight into the mechanism of regulated delivery of integral membrane proteins to the plasma membrane. Recent work in Dr. Kaiser's laboratory has shown that in yeast delivery of the general amino acid permease (Gap1) to the plasma membrane responds to the nitrogen source in the growth medium. The regulation of the cellular location of Gap1 takes place in the trans-Golgi where Gap1 is loaded into transport vesicles directed either to the plasma membrane or to the vacuole. In this application, Dr. Kaiser proposes to elucidate the mechanism of Gap1 sorting in the trans-Golgi. These studies will include development of in vivo and in vitro assays for the formation of Gap1-containing vesicles, and the identification and characterization of gene products that control Gap1p sorting. The regulated delivery of proteins to the plasma membrane is key to human cell physiology. For example, the GLUT4 glucose transporter is delivered to the plasma membrane of fat and muscle cells in response to insulin, and defects in this regulated trafficking are though to be a root cause of non-insulin- dependent diabetes. Little is known of the mechanisms that control the location of GLUT4 or other regulated membrane proteins in humans. However, in S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to uncover the genes and proteins responsible for regulated sorting in the Golgi. The genes from S. cerevisiae should give access to the mammalian genes that perform similar functions, opening the way to new methods for the diagnosis of dysfunctional sorting in mammalian cells and providing new opportunities to selectively alter the plasma membrane composition of cells.

DESCRIPTION (provided by applicant): The activities of integral membrane proteins, such as transport proteins, are often regulated by intracellular sorting. Such sorting process can produce rapid changes in the rate that a transporter is delivered to the plasma membrane in response to an intracellular signal and thus provide a way for a cell to alter its capacity to take up small molecules from the extracellular environment in response to regulatory cues. For example, the GLUT4 glucose transporter is delivered to the plasma membrane of fat and muscle cells in response to insulin, and defects in this regulated trafficking are thought to be a root cause of non insulin-dependent diabetes. Many aspects of the intracellular sorting of GLUT4 remain poorly understood at this time. Dr. Kaiser's research group proposes to continue to study regulated sorting of general amino acid permease (Gap1) in response to the nitrogen source in the growth medium. By studying amino acid permease sorting in S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to elucidate the mechanisms responsible for regulated sorting in the late secretory pathway. Work in the previous funding period has identified a large set of genes that control the sorting of Gap1. These genes include factors directly involved in the membrane trafficking of Gap1, such as proteins required to modify Gap1 with poly-ubiquitin, a tag found to be required for the proper sorting between Golgi and endosomal compartments. In addition, genes required to generate the proper regulatory signals for Gap1 sorting were also identified. Building on these findings the proposal is to: (i) determine how Gap1 is ubiquitinated and to identify the cellular components required for recognition of the ubiquitin tag, (ii) use a combination of genetic and biochemical methods to characterize the gene products that control Gap1 sorting and to determine their site(s) of action in the secretory and endocytic paths, and (iii) determine how nitrogen-derived signals are generated and determine how the membrane trafficking machinery responsible for sorting Gap1 decodes these signals.

[unreadable] DESCRIPTION (provided by applicant): The activities of integral membrane proteins, such as transport proteins, are often regulated by intracellular sorting. Such sorting processes can produce rapid changes in the rate that a transporter is delivered to the plasma membrane in response to an intracellular signal and thus provide a way for a cell to alter its capacity to take up small molecules from the extracellular environment in response to regulatory cues. For example, the GLUT4 glucose transporter is delivered to the plasma membrane of fat and muscle cells in response to insulin, and defects in this regulated trafficking are thought to be a root cause of non insulin-dependent diabetes. Many aspects of the intracellular sorting of GLUT4 remain poorly understood at this time. Dr. Kaiser's research group proposes to continue to study regulated sorting of general amino acid permease (Gap1) in response to the nitrogen source in the growth medium. By studying amino acid permease sorting in the model organism S. cerevisiae it will be possible to elucidate the mechanisms responsible for regulated sorting in the late secretory pathway. Work in the previous funding period has shown that Gap 1 p sorting is controlled by the rate at which Gap1 p protein can recycle from the endosome to the plasma membrane and that this sorting step is controlled by the abundance of intracellular amino acids. A genetic screen for mutants defective in Gap1 p recycling identified a GTPase containing complex (GSE) that interacts with Gap 1 p so as to suggest that the GSE complex may form part of a vesicle coat for Gap 1 p trafficking. Building on these findings the proposal is to: (i) identify additional components of the GSE complex with the ultimate aim of reconstituting activity in vitro, (ii) determine the structure and function of the GTPase component of the GSE complex, including functional studies of the mammalian orthologs, and (iii) determine how the nitrogen source controls Gap1 p recycling. We have found that Gap1 p modification by ubiquitin is necessary for sorting from the Golgi to endosome. A final aim of the project is (iv) to understand how ubiquitination is regulated and to identify the cellular components required for recognition of the ubiquitin tag. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is the compartment where membrane and secretory proteins are modified, folded, and assembled. A key step in the folding of most extracellular proteins includes formation of disulfide bonds, which can add stability to folded polypeptides and can link together subunits of protein complexes. Previous work on this project has delineated the core pathway for protein disulfide bond formation in eukaryotic cells in which disulfide bonds generated by an oxidase, Ero1p, are then transferred to protein disulfide isomerase (PDI), which in turn transfers a disulfide bond to substrate proteins. Through a combination of biochemical, structural, and genetic experiments in the yeast S. cerevisiae we have gained fundamental insight into the molecular mechanisms that underlie each of the steps in this pathway. In this application we now focus on understanding how the protein oxidation pathway is integrated into the redox biochemistry of the cell. Aims of the proposal include: understanding how Pdi1p and related proteins regulate Ero1p activity, investigation of a novel block in ER translocation instigated by hyperactive Ero1p activity, and elucidation of a new electron transport chain in the ER coupled to disulfide bon formation. PUBLIC HEALTH RELEVANCE: In S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to uncover the genes and proteins responsible protein folding in the ER. A detailed understanding of these pathways in S. cerevisiae will make it possible to understand parallel processes in mammalian cells, opening the way to diagnose dysfunctional folding in the ER of mammalian cells and possible detrimental effects of reactive oxygen species generated by the disulfide bond formation pathway.

StarCellBiology employs a set of in silico cell biology experiment simulations for use in generating realistic problems for undergraduate student lecture and laboratory classes. The cell biology simulations will augment the biochemistry (StarBiochem) and genetics (StarGenetics) materials already on the STAR web site http://web.mit.edu/star/.

Intellectual Merit: The materials and accompanying faculty guidelines are enriching science education by supplying educational software and curricular materials that (i) provide for inquiry-based learning experiences through interactive, realistic, computer-generated simulations of experiments, (ii) expose students to examples of actual data acquired from cutting-edge research, (iii) are useful across a range of educational levels, (iv) are designed to accommodate instructor-determined customization, (v) can be easily implemented in existing course curricula, and (v) are freely and openly accessible worldwide via the internet. This interactive, inquiry-based, virtual experiment simulator enables students to engage in the experimental reasoning process by providing virtual opportunities for them to determine what experiments are needed to answer particular biological questions, design and perform those experiments, analyze the data generated, perform follow-up experiments, and draw conclusions from their own inquiries. It offers students the opportunity to interact with real visual cell biology data in the course of their studies, as well as utilize microscopy image and time-lapse movie data in demonstrations of key topics, including cell cycle, intracellular localization and colocalization, trafficking, signaling, cell motility, cell diversity, and tissue organization.

Broader Impact: Following a design model successfully employed in the two sets of STAR materials currently available, the specific components of each StarCellBio experiment simulation are defined by the instructor through use of a modifiable template that is freely available online, and a beta version of StarCellBio was assessed for function and usability by faculty and students. The evaluation plan utilizes research instruments well established in science education and includes mixed-method approaches to (i) measure gains in student knowledge and comprehension of cell and molecular biology concepts, (ii) probe development of student ability to design, conduct, and reasonably interpret results of biological experiments, (iii) measure changes in students' attitudes and motivation towards pursuing further study in biology, and (iv) query overall student response to the StarCellBio tool. Broad dissemination approaches include online advertising campaigns and direct mailings to biology faculty at universities nationwide.

This project is being jointly funded by the Directorate for Biological Sciences, Division of Biological Infrastructure and the Directorate for Education and Human Resources, Division of Undergraduate Education as part of their efforts toward Vision and Change in Undergraduate Biology Education.