Peter Novick - US grants

Yeast cells, like most eukaryotic cells, exhibit cell polarity. The polarized growth of yeast depends upon the localized incorporation of new surface components into the region of the cell known as the bud. This is mediated by the fusion of Golgi-derived secretory vesicles with this specialized region of the plasma membrane. We have defined three systems that work together to mediate the targeting, docking and fusion of secretory vesicles with specific regions of the yeast plasma membrane. Sec2 and the GTP-binding protein Sec4 are needed for the polarized concentration of vesicles at fusion sites. Sec3, Sec 5, Sec 6, Sec 8, Sec 10, Sec 15 and Exo7O comprise the components of a complex, termed the Exocyst, that may specify regions of the plasma membrane that are active in vesicle docking. Sec1 and Sec9 may control the interaction of Snc (an integral protein of.the vesicle) with Sso (an integral protein of the plasma membrane) to complete the transport event We will analyze interactions among the components of each system and between the different systems. In specific: 1) We will use genetic, biochemical and electron microscopic approaches to better define the structure of the Exocyst 2) We will determine which Exocyst subunit mediates membrane attachment, and identify the Exocyst receptor on the plasma membrane 3) We will test the hypothesis that the Exocyst functions to dock vesicles, and that this activity is regulated by the GTP-bound form of Sec4 on the vesicle. 4) We will determine if different sec mutants block the pathway before or after formation of the Snc-Sso complex. 5) We will analyze the phenotype of recessive and dominant SnC mutants and define the functions of their high copy suppressors. 6) We will define the interactions of Sec l with the other components of the exocytic machinery.

The overall goal of this Program Project is to understand the molecular mechanisms involved in the control of protein and membrane traffic in normal and transformed cells. It consists of seven individual projects: Project 1: The goal of this project is to characterize Golgi subcompartments and to identify functionally important membrane proteins found in these subcompartments. The trafficking of the lysosomal Man-6-P receptor (46 Kd) through Golgi subcompartments will also be investigated in normal and transformed cells. Project 2: This project takes advantage of the genetic approach and the availability of mutants defective in specific steps along the secretory pathway to identify proteins or regions of proteins that are important in targeting along the exocytic pathway. One such mutant to be investigated has a defective gene that appears to be a member of the ras oncogene family. Project 3: The goal of this project is to investigate the role of protein conformation on signaling transport along the secretory pathway using viral membrane proteins as models. Project 4: This project will investigate the role of the binding protein BiP in post-translational processing and ER to Golgi transit of nascent secretory and membrane proteins in normal and transformed cells. Project 5: The purpose of this project is to determine the mechanisms of sorting and traffic control during endocytosis using closely related Fc receptors in normal and transformed cells expressing this receptor from clone cDNAs. Project 6: The goals of this project are to define the molecular basis for the transient failure of the membrane protein, glycophorin, to be segregated in the ER in transformed Friend Erytholeukemic cells, and to attempt to identify sorter proteins and carrier proteins involved in control of vesicular membrane traffic. Project 7: Hybrid proteins generated by gene fusion and expression technologies have been shown to be blocked at various steps along the biosynthetic pathway. In this project the precise site of the blockage and the molecular features responsible for the blockages will be investigated.

Function of the Amphiphysin Protein Family project I will address the role of amphiphysin and interacting proteins, dynamin, synaptojanin and AP2 in endocytosis, cell polarity and cell proliferation. Changes in the expression and function of amphiphysin isoforms and amphiphysin- interacting proteins during normal growth and following the loss of growth control it will be determined. Ypt51 Interacting Components in Endocytosis: Project II focuses on the role of Ypt51 and interacting proteins in yeast endocytosis. Functional interactions with fibrin and amphiphysin homologs indicate a role of the cortical cytoskeleton. Additional interacting genes have been identified through a synthetic lethal screen. The relationship between these gene products and Ypt51 function on the endocytic pathway will be defined. Regulated Exocytosis in Developing and Neoplastic Pancreatic Acinar Cells: Project IV concerns the role of rab proteins in the developing rat pancreas. During development, secretogenesis correlates with the movements of rab3D from the cytosol to the granule membrane and the transient appearance of rab4 on the actin terminal web. The changes in rab localization, function and protein-protein interactions will be determined. Control of Cell Polarity and Plasma Membrane Function by rho Family GTPases: Project V will explore the development and maintenance of cell polarity in epithelial cells, neurons and lymphocytes. The role of the rho family of GTPases and of mammalian homologs of yeast genes involved in polarized growth will be addressed. The changes in localization and function of these proteins in metastatic cells will be defined. Sec4 and Interacting Proteins in Cell Polarity: Project VI focuses on the contribution of Sec4 and interacting proteins to polarized cell growth in yeast. Polarized concentration of secretory vesicles depends upon Sec2, actin and a myosin. Sec 2 localizes to sites of exocytosis and directly interacts with Sec4 in a nucleotide dependent fashion. This project will define the role of Sec2, Sec4 and the cytoskeleton in polarized growth. GROWTH=P01CA461280008 The long term goal of this proposal is to improve our understanding of the properties of the cortical cell cytomatrix and of its pleiotropic effects on a variety of cell processes including endocytosis and cell proliferation. More specifically, this grant application proposes to elucidate the role of the amphiphysin/RVS family which is compromised of at least two members in man, amphiphysin I and II, and with two members in yeast, Rvs161 and Rvs167. The RVS yeast genes have been implicated in endocytosis, cell polarity, entry into stationary phase and actin dynamics. Amphiphysin I is expressed primarily by neurons, is concentrated in the peripheral cytomatrix of nerve terminals, and has a putative role in endocytosis via its interaction with the GTPase dynamin I, the inositol-5-phosphatase synaptojanin and the clathrin adaptor AP2. Amphiphysin II undergoes extensive regions of the subplasmalemmal cytoskeleton of muscle and nerve cells. Both amphiphysin I and II, like other proteins of the cortical cytoskeleton, have been linked to cancer. Amphiphysin I is ectopically expressed in some cancer cells and is a dominant autoantigen in Stiff-Man syndrome associated with breast cancer. Amphiphysin II was found to interact with the oncogene Myc and to be a tumor suppressor gene. The working hypothesis of this proposal is that proteins of the amphiphysin family are general components of the subplasmalemmal cytoskeleton, and that the pleiotropic actions of the amphiphysins reflect the multiple functions of the cortical cell cytoplasm. This application proposes 1) to perform a thorough characterization of the amphiphysin protein family in neurons, muscle and epithelial cells, 2) to identify interacting partners of amphiphysin isoforms and to study the functional role of these interactions (with emphasis on endocytosis, actin dynamics and cell proliferation) by using biochemical experiments, cell free assays, microinjection experiments, cell transfections, 3) to determine the phenotypic changes in mice caused by manipulations of the amphiphysin genes, 4) to investigate changes in amphiphysin expression in human cancer tissues and to establish whether these changes have a pathogenic role in some form of cancer. These studies will provide new important information on functional aspects of the cell periphery which are highly conserved among all eukaryotic cells, and may offer new insights into the pathogenesis of some forms of cancer, providing clues toward their early diagnosis and therapy.

The great diversity of techniques used in Cancer Research demands equally diverse reagents and facilities. While the preparation of growth media is not technically difficult, the scale of our needs requires an efficient centralized facility. The media preparation needs for the Program will be provided by the Media Preparation Facility. This facility will prepare both liquid and solid media for the growth of bacteria and yeast, plus tissue culture media, salts, and buffers. In addition, sterile glassware and supplies will be provide for the Projects in the Program.

Sec4 is a small GTPase of the rab family that is associated with the secretory vesicles of yeast. Sec 4 interacts with different sets of proteins in its different nucleotide states. In its GDP bound state it interacts with GD1, a proteins that maintains Sec4 in the cytosol. In its nucleotide free state, Sec4 will interact with DSS4 or with Sec2. Dss4 acts to stimulate dissociation of nucleotides for Sec4, while Sec2, working in conjugation with the actin based cytoskeleton, is needed for the polarized concentration of secretory vesicles at sites of exocytosis. Fst1 is a proteins that interacts with Sec2 to negatively regulate its function. Several newly identified proteins will interact with Sec4 in its GTP bound state. A number of studies are proposed to address the mechanisms by which these proteins work together to control the cycle of Sec4 function and to allow it to fulfill its role in targeting vesicles to specialized regions of the cell surface. 1) We will define the structural domains of Sec2 that allow binding to Sec4, oligomerization, localization to the bud tip and interaction with Fst1. 2) We will determine the mechanism by which Sec2 is localized to the bud tip and identify factors that interact with its localization domain, possibly linking it to the cytoskeleton. 3) We will establish the role of Sec2 in Sec4 function, evaluating its effect on the GDP off rate, the GTP on rate and the interactions with Dss4, Gdi1 and the cytoskeleton. 4) We will analyze the role of two different suppressors of dominant negative alleles of SEC4;SMY1 and a novel gene encoding 27kD protein. 5) We will explore a novel approach to identify a GAP for Sec4 and pursue the function of several Sec4 interacting proteins and a highly conserved protein that interacts with Dss4.

protein protein interaction; exocytosis; biomedical resource; secretion;

protein localization; transport proteins; protein structure function; biomedical resource; secretion; biological transport;

intermolecular interaction; biomedical resource; biological transport;

DESCRIPTION (provided by applicant): In all eukarotic cells, the endoplasmic reticulum (ER) forms a highly fenestrated tubular network that spreads throughout the volume of the cell, often lying just below the plasma membrane. This distribution may be critical for calcium signalling and other important cellular functions. We have taken a genetic approach towards understanding the mechanism of ER dynamics, structure and inheritance in yeast. Our work to date shows that ER tubules form from the nuclear envelope at the start of the cell cycle, they are delivered into the bud along actin cables by the type V myosin, Myo4p, they are anchored at the bud tip by the exocyst complex and then spread along the cell cortex to form the cortical ER. We propose that Rtn1 p, a member of the conserved reticulon family of proteins, functions as the receptor for the exocyst subunit Sec6p on the ER membrane and that Rtn1 p also serves to link the ER to the cell cortex. Four specific aims are planned: 1) We will analyze the interaction of Rtn1p with the exocyst, defining the topology of Rtn1p within the ER membrane and the specific interactions that link the exocyst to Rtn1p. 2) We will determine if Rtn1p binds to the Tcb3 protein along the cortex and if this interaction underlies the unique localization of Rtn1p as well as the tight association of the ER with the cell cortex. 3) We will determine if Rtn1p plays a role in forming the fenestrated reticulum characteristic of the ER in all eukaryotes and if it interacts with other components of the ER membrane in this capacity. 4) We will express or microinject dominant negative alleles of mammalian reticulon proteins as well as make use of RNAi methodology to inhibit the function of the four mammalian RTN proteins. Phenotypic analysis will determine if the roles of Rtn1p and its interactions are conserved.

[unreadable] DESCRIPTION (provided by applicant): Membrane traffic is required for a broad range of essential cellular functions, such as controlling the accessibility of cell surface receptors, the translocation of glucose transporters in response to insulin, antigen presentation, neuronal transmission and the establishment and maintenance of epithelial cell polarity. Therefore, the regulation of membrane traffic is directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. Rab GTPases are key regulators of membrane traffic. By recruiting and activating a functionally diverse set of effectors, a single Rab GTPase can coordinate the various sub- reactions within a given stage of membrane traffic, including vesicle budding, delivery, tethering and fusion. Furthermore, our results indicate that adjacent stages of transport can also be coupled through coordinated rab regulation. We recently defined a rab guanine nucleotide exchange factor (GEF) cascade in which one rab, in its GTP-bound state, recruits the GEF that activates the next rab along the exocytic pathway. We also have preliminary evidence for a rab GTPase activating protein (GAP) cascade operating in a counter current fashion. Here the downstream rab recruits the GAP that inactivates the upstream rab. The net effect is rab conversion in which a given patch of membrane starts out labeled with one rab, but over time becomes labeled with another rab. Since each rab recruits and activates a distinct set of effectors, this leads to a functional maturation of the membrane. We will explore these two cascade mechanisms in further detail and test the physiological consequences of uncoupling adjacent stages of membrane traffic. We will test the role of a Sec4p effector in SNARE assembly and explore the roles of several new putative Sec4p effectors. Through these studies we will begin to define the exocytic pathway as a fully coordinated system, rather than as a collection of isolated sub-reactions. Membrane traffic is the mechanism by which material is transferred between different compartments within the cell and the regulation of membrane traffic is directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. This study addresses the molecular mechanisms by which different stages of membrane traffic are coordinately regulated. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Rabs represent the largest branch of the Ras GTPase superfamily, with ten members in yeast and more than 60 in mammalian cells. They serve as master regulators of membrane traffic, each typically controlling several different aspects of a specific stage of membrane traffic by recruiting diverse effectors proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange proteins (GEFs) that catalyze the displacement of GDP and binding of GTP and GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. Recent work from our lab has demonstrated that different Rabs are networked to one another through their regulators. Specifically we have shown that the Rab, Ypt32, in its GTP-bound form recruits Sec2, the GEF that activates the downstream Rab, Sec4, as well as Gyp1, the GAP that inactivates the upstream Rab, Ypt1. The net effect is a programmed series of Rab conversions that lead to changes in the functional identity of the membrane as it flows along the exocytic pathway. We have also shown that the Golgi pool of phosphatidylinositol 4-phosphate (PI4P) works in concert with Ypt32 to recruit Sec2 and to control a regulatory switch in Sec2 function. We propose five specific aims to address the molecular mechanisms by which Rabs are networked to one another and by which the distribution of PI4P is spatially defined. 1. We will explore the role of phosphorylation in the regulation of Sec2 function. Phosphomimetic and non- phosphorylatable alleles will be tested for their interactions with Sec4, Ypt32, Sec15 and PI4P. 2. The Sec4 GEF Sec2 binds to the Sec4 effectors Sec15 leading to a positive feedback loop. To test the role of this mechanism in membrane traffic we will generate Sec2 alleles specifically defective in Sec15 binding. 3. We have generated an allele of Ypt1 that can be activated by the Sec4 GEF, Sec2. We will determine if this mutation redirects the membrane association of Ypt1 and the effects of this short circuit on membrane traffic. 4. We have evidence for a second example of a Rab-GAP cascade and will screen all Rab GAPs against representative Rabs to identify more. We will test the effects of GAP disruption on overlap of Rab domains. 5. PI4P is normally restricted to the Golgi and is not enriched on Golgi-derived secretory vesicles. We will determine the mechanism by which PI4P is limited to the Golgi.