Tom Hall Stevens - US grants

This proposal examines the very basic cell biological questions of how eukaryotic cells sort proteins within the Golgi body and separate out those proteins destined for the lysosome, and ultimately deliver these proteins to the lysosome. The simple eukaryote yeast will be used as a model system, since the secretory pathway in yeast has been found to be strikingly similar to that observed in higher eukaryotic organisms. Yeast offers the advantage that it is readily amenable to genetic and biochemical analysis. Yeast mutants will be obtained that are defective in the sorting, packaging and transport of glycoproteins to the lysosome-like vacuole. These mutants will be analyzed to determine precisely what step in the vacuole assembly pathway is defective. In conjunction with studying the components of the vacuole assembly pathway, the recognition of sorting signals present on the vacuolar proteins that target their localization will be investigated. These studies should provide molecular information on the nature of eukaryotic protein recognition signals and how these signals control the intracellular localization of proteins. The diseases pseudo Hurler polydystropy and l-cell disease arise because of genetic defects preventing the correct localization of lysosomal enzymes. In addition, defects in the basic process of secretion may be relevant to the disease cystic fibrosis.

The overall goal of this research is to develop a mechanistic understanding of the vacuolar biogenesis pathways and endosome function in the yeast Saccharomycescerevisiae. Genetic analysis has led to the identification of a very large number of components required along the vacuolar assembly pathway in yeast, and two of these proteins, Vps55 and Vps68 are particularly of interest because these are integral membrane proteins that function at the endosome. Vps55 and Vps68 form a complex together with UipSp, a 25 kDa member of the large DUP240 gene family. The role of this integral membrane protein complex is being investigated as a docking/tethering site for the large collection of soluble Vps proteins that are recruited to the Golgi and endosome membrane during protein sorting and vacuole membrane traffic. "Class E" proteins proteins function at the endosome in the formation of the multi-vesicular body (MVB) and the sorting of cargo proteins into these intralumenal vesicles. Ten of these Vps proteins form three separate ESCRT protein complexes (Endosome Sorting Complex Required for Transport), and the AAA- ATPase Vps4 is required to disassemble the ESCRT complexes. We are investigating the role of three other Class E Vps proteins that are required to recruit the Vps4 complex to the endosome and to stimulate ESCRT disassembly by activating the Vps4 ATPase activity. We will also exploit our new genetic screen for mutants defective for membrane invagination at the endosome;analysis of these genes should reveal important insight into the mysterious process of MVB membrane invagination. We are investigating the connection between endosome function and the ability of yeast to sense glucose in the extracellular environment. Some of the yeast Class E mutants are Sucrose NonFermenting (Snf-), and only a subset of each of the ESCRT complex polypeptides are required for yeast to be Snf+. The high-affinity glucose sensor in yeast, Snf3, localizes to MVBs in yeast mutants defective for MVB function. Studies of membrane traffic in yeast have resulted in a deeper understanding of membrane transport in all eukaryotic cells, and these studies in yeast are also providing important insights into our understanding of lysosomal storage diseases and the cellular requirements for HIV viral particle formation.

The work is aimed at understanding the sorting and transport of membrane proteins to the yeast lysosome-like vacuole. The simple eukaryote yeast will be used as a model eukaryotic sorting system, since the secretory and vacuole assembly pathways are very similar to the pathways in animal cells. Studies in yeast offer a unique opportunity to investigate the complex processes involved in membrane protein sorting and transport by taking advantage of the ability to exploit the powerful genetic approaches available in yeast. It also appears likely that the basic cellular functions that facilitate sorting of vacuolar/lysosomal membrane proteins will be conserved across all eukaryotic cells. Yeast mutants that mislocalize the vacuolar membrane protein dipeptidyl amino-peptidase (DPAP-B) will be obtained by exploiting a newly developed selection procedure. These mutants will be screened biochemically and by immunogold labeling for the secretion of a large number of soluble and membrane-bound vacuolar proteins. In addition, a major effort will be made towards identifying the vacuolar sorting and transport signals present on the membrane protein DPAP-B. Mutations in the DPAP-B structural gene will be generated and those resulting in missorting of enzymatically active DPAP-B will be identified with the DPAP-B mislocalization selection procedure. The structural genes of the two largest subunits of the yeast vacuolar membrane H+-translocating ATPase will be cloned using the Lambdagtll yeast library. Mutations will be constructed in these genes to elucidate the role of this H+-ATPase in acidification of the vacuole. These mutations will also permit an analysis of the role of acidification in the sorting of newly synthesized vacuolar hydrolases, in fluid-phase and receptor-mediated endocytosis, and in the function of the vacuole. The biosynthesis, assembly, targeting and transport of this vacuolar multi-subunit membrane-bound H+-ATPase complex will be investigated in an effort to understand the relationship between the synthesis and assembly of the subunits and their transport to the vacuole. These studies are likely to increase our basic understanding of diseases that result from missorting of lysosomal hydrolases such as Mucolipidosis II and III and other lysosomal storage diseases.

The overall goal of this research is to develop a mechanistic understanding of membrane protein sorting and assembly in the yeast Saccharomyces cerevisiae. Recent results indicate that the vacuole is the default compartment for membrane proteins that enter the yeast secretory pathway. To explain these results the vacuolar default model has been proposed. This model states that membrane proteins of the ER and Golgi contain retention signals preventing their transport to the vacuole, whereas plasma membrane proteins have positive sorting information, which ensures that they are not transported to the vacuole. The retention signal on the yeast Golgi membrane protein DPAP A has been characterized, and this information will be used to design genetic screens to identify components of the yeast Golgi retention apparatus. Specific aims #1 and 2 are focused on the identification of components required for retention of membrane proteins in the yeast Golgi, and the molecular genetic and biochemical characterization of these components. Yeast mutants will be isolated that fail to retain a Golgi membrane protein, and the GAL4 two-hybrid genetic system will be used to identify genes encoding proteins that directly interact with the DPAP A Golgi retention signal. The genes identified will be sequenced, disruption alleles constructed to test the in vivo function of the components, and the Golgi retention machinery biochemically characterized. Specific aim #3 will test the second part of the vacuolar default model by investigating the nature of the targeting information that directs the FUS1-encoded plasma membrane protein to the yeast cell surface. Gene fusions between the FUS1 gene and the gene encoding the yeast Golgi membrane protein, KEX1, will be constructed to map any region of the FUS1 protein capable of redirecting KEX1 protein lacking its Golgi retention signal to the yeast plasma membrane. A second focus of this proposal is the mechanistic analysis of the assembly and targeting of a multisubunit membrane protein complex, the yeast vacuolar membrane H+-ATPase. With a majority of the vacuolar H+- ATPase subunit genes cloned, the tools are now available to begin to address issues of the biosynthesis of this complex vacuolar membrane protein. Specific aim #4 is designed to isolate and characterize vacuolar H+-ATPase deficient yeast mutants (vma), clone and characterize the VMA genes, and to investigate the function of the VMA proteins. We will focus on the VMA6, VMA21, and VMA22 genes and their encoded proteins, since these factors are absolutely required for the assembly of both the membrane and peripheral sectors of the yeast vacuolar H+- ATPase complex. The final specific aim (#5) is focused on investigating the biosynthesis, assembly, and targeting of the yeast vacuolar H+-ATPase membrane complex. The roles of individual VMA proteins in the assembly and function of the membrane sector of the vacuolar H+-ATPase will be assessed. The long-term objective of the studies with the vacuolar H+- ATPase is to develop a mechanistic understanding of the pathway of assembly for this very complex membrane protein.

9317993 Dahlquist Partial funding of the hardware needed to perform pulsed field gradient experiments to enhance the use of nuclear magnetic resonance to study the solution properties of proteins and nucleic acids has been requested. The requested capability will greatly aid in 8 projects outlined in this proposal. These include investigations of the substrate binding site of galactose oxidase, the solution structure of the SYMBOL 101 \f "Symbol" subunit of the mitochondrial ATP synthase, the structure of an unusual DNA binding domain found in the project of the skn-1 gene of C. elegans, the structural basis of the interaction of two yeast transcription factors a1 and a2, the structural changes associated with mutations of the prosequence of the yeast carboxypeptidase and the folding pathway of the heterodimeric bacterial luciferase. s t g ? r StringFileInfo ^ 040904E4 ' CompanyName Microsoft Corporation : & FileDescription Windows Task Manager app9317993 Dahlquist Partial funding of the hardware needed to perform pulsed field gradient experiments to enhance the use of 4 5 K L o q $ $ $ D Y Y ? CG Times Symbol & Arial Tms Rmn Times New Roman Y 9 A A " h B e ~ < 1 Shauna Benson Deseree King, BIR

An Affinity sensors IAys Optical Biosensor will be purchased for studies in assembly and regulatory reactions in replication and transcription, the energetics of assembly of the bacterial chemotaxis signaling complex, the role of SNARE proteins in controlling the specificity of membrane fusion in yeast, assembly and structure of phage and transcription complexes, assembly and function of the RNA polymerase II transcription complexes and the role of chaperones in protein folding.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this application to the NCRR Shared Instrumentation Program is to request funds for the purchase of an LCQ Deca XP ion trap mass spectrometer, fully integrated and optimized for proteomics. This ProteomeX Workstation will be an important addition to our newly formed Genomics and Proteomics Facility (GPF), which is a core facility serving the Biosciences Group at the University of Oregon. The Biosciences Group is made up of researchers from the Institute of Molecular Biology, the Institute of Neuroscience, and the Molecular Evolution Group. Mass spectrometry has become an essential tool in the characterization of proteins and macromolecular complexes, and has become a particularly powerful tool with the completion of the genome sequences for a wide variety of organisms, such as human, yeast, fruit fly, C. elegans, Neurospora, zebra fish, as well as a large number of bacterial species. Twelve major research groups have described specific projects that will benefit from the use of the LCQ and, more specifically, LC/MS/MS analysis of complex mixtures of proteins. The major users will be: Tom Stevens, Roderick Capaldi, Frederick Dahlquist, Brian Matthews, Chris Doe, Alice Barkan, George Sprague, Beatrice Darimont, Bruce Bowerman, Eric Selker, as well as two of our newest faculty Ken Prehoda and Andy Berglund. Minor users will include: Peter von Hippel, Jim Remington, Bill Roberts, Diane Hawley, Eric Johnson, and Karen Guillemin. The sighting of this mass spectrometer at the University of Oregon will make this technology immediately accessible to our researchers, rather than having to rely on long distance collaborations. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Continued support is requested for an ongoing program of graduate research training in molecular biology and biophysics at the University of Oregon. This training activity is centered in the Institute of Molecular Biology, and also involves additional groups with related scientific interests. Funds are requested for 12 predoctoral positions, within a program that includes approximately 59 graduate students, 47 postdoctoral trainees, and 22 training faculty. The program places special emphasis on the control of gene expression and nucleic acid structure/function, molecular basis of signaling and cell function, protein structure and dynamics, and molecular approaches to development and differentiation. The basic aspect of the training is laboratory research carried out under the direction of a facult member in the molecular biology and biophysics training program. Through this experience, the trainee becomes skilled at posing questions about fundamental biological processes and designing experiments to answer those questions. The training is augmented by formal courses offered by the Biology, Chemistry, and Physics Departments, by seminar programs that highlight current research in molecular biology, biophysics, and related disciplines, by the close involvement of a Thesis Advisory Committee, and by research seminar and journal club presentations by trainees. The training facilities include the laboratories of the faculty and support services such as the structural biology biophysical facility, the state-of-the-art genomics and imaging facilities, and a large number of other modern facilities. Major equipment is shared and housed in common space. The laboratories of most of the faculty are contiguous and in interconnected buildings. This arrangement fosters strong interdisciplinary interactions and collaborations among faculty and students.