Suzanne R. Pfeffer - US grants

The sorting of proteins to lysosomes is studied because this is presently the best understood sorting process. Upon arrival in the Golgi complex, newly synthesized lysosomal enzymes acquire a mannose 6-phosphate (man6P) moiety that enables these enzymes to bind to man6P-specific receptors. The man6P receptor-enzyme complexes are then delivered by transport vesicles to an acidic prelysosome, where the enzymes are released; the man6P receptors then return to the Golgi complex for another round of transport. The biochemistry of this delivery process is being studied by reconstituting the vesicular transport of the man6P receptor from prelysosomes back to the Golgi complex in a cell free system. Now the project will define the molecular components that are used by the cell to form transport vesicles, target them to a specific destination, and enable them to fuse. Since almost nothing is known about the molecular components that facilitate intracellular transport, it may be possible to identify new proteins that mediate this vesicular transport process. To streamline operations, eukaryotic cells have devised a set of compartments that carry out specialized tasks, and therefore must be supplied with a unique set of proteins. The goal of this research is to understand, in molecular terms, how cells establish and maintain distinct membrane-bound compartments. This will lead to an understanding of the molecular mechanisms by which proteins are targetted to specific intracellular estinations.

A fundamental question in cell biology is how organelles achieve their distinct cytoplasmic localizations. In interphase cells, the Golgi complex is found in close proximity to the microtubule-organizing center (MTOC), on one side of the nuclear envelope. During mitosis, the golgi vesiculates into smaller units which disperse throughout the cytoplasm. At telophase, the vesicles coalesce and return to a pericentriolar location. The long term goal of this research is to elucidate the protein-protein interactions responsible for the specific and cell cycle- regulated, subcellular localization of the Golgi complex. We have shown that isolated Golgi complexes can interact with cytoplasmic components of semi-intact cells, and accumulate in the vicinity of the centrosome. This process requires ATP hydrolysis, the presence of intact microtubules, and is entirely dependent upon the microtubule-based motor, cytoplasmic dynein. The specific aims of the present proposal are to isolate and characterize a potentially novel factor that we have shown is essential for Golgi accumulation at the centrosome. This factor can be released from Golgi membranes by KCI treatment. We wish to determine if this protein acts as a linker to couple the Golgi complex to dynein, and/or microtubules, in a cell-cycle-regulated fashion. This factor has the potential to represent a new class of proteins that can link a molecular motor to a specific membrane-bound organelle.

molecular biology; membrane; meeting /conference /symposium; membrane proteins;

The long term goal of this research is to understand the molecular basis of receptor trafficking in mammalian cells. The goal of this proposal is to study how one type of receptor is collected into a transport vesicle and delivered to another subcellular compartment. Specifically, we seek to understand the mechanism by which a newly discovered protein, TIP47, facilitates the collection of mannose 6-phosphate receptors into transport vesicles that bud from endosomes and carry the receptors to the trans Golgi network. TIP47 recognizes a phenylalanine/tryptophan signal in the cytoplasmic domain of the cation-dependent MPR that is essential for its retrieval from endosomes and delivery to the Golgi. We describe here experiments designed to investigate why mannose 6- phosphate receptors are recognized by a specific trafficking machinery depending upon their intracellular localization. We also propose to continue our analysis of the Rab9 GTPase in terms of how it regulates MPR trafficking. Rab GTPases are believed to function in vesicle docking. We have discovered a novel Rab9 effector, p40, which binds preferentially to the active, GTP-bound form of Rab9. p40 is a highly active transport factor. We propose experiments designed to determine if p40 functions in vesicle docking. These studies have important implications for our understanding of growth control and antigen processing, and will provide fundamental information regarding the mechanism of receptor trafficking in mammalian cells.

DESCRIPTION (provided by applicant): The long term goal of this research is to understand the molecular basis of protein trafficking between membrane-bound compartments in human cells. Protein transport involves cargo collection into vesicles, vesicle budding, motility, tethering and docking at the target membrane, and subsequent fusion. Tethering and docking are the least understood steps in membrane traffic. The specific goal of this application is to investigate the molecular function of a protein named GCC185, a 185K, trans Golgi network (TGN)-localized protein of the GRIP domain family. We seek to test the hypothesis that GCC185 functions as a tethering protein for transport vesicles arriving at the TGN. With regard to GRIP domain-family Golgins, the most important questions that need to be resolved are: 1. Are these proteins actually tethers? 2. What molecules do these proteins partner with to achieve tethering? 3. Do these proteins act as vesicle-associated tethers or target-associated tethers? 4. Do these proteins bind to TGN-specific SNARE proteins, and how are they released from the Golgi during or after vesicle fusion? To begin to address these questions, we propose to: 1. Use biochemical approaches to determine the molecular basis for GCC185 Golgi complex localization;2. Test a model for GCC185 as a vesicle-bound tether by accumulating transport vesicles in SNARE- depleted cells;3. Characterize the binding of GCC185 to SNARE proteins implicated in endosome to Golgi transport, and test whether GCC185 catalyzes SNARE complex formation at the TGN;4. Establish a membrane tethering assay using purified, immobilized GCC185 to explore its function. This is important because it will establish that GCC185 is a bona fide tethering protein, and may help us to purify these transport carriers for the first time. These experiments will provide important clues to the mechanism by which GCC185 is localized to the trans Golgi network, and what this protein does there, to facilitate the docking and fusion of transport vesicles, inbound from late endosomes. This work has broad implications for our understanding of vesicle docking and fusion events within the secretory and endocytic pathways that are essential for normal human health and disease.

Project Summary/Abstract The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. Several recently discovered proteins are needed for MPR transport from late endosomes to the trans Golgi network: a cargo selection protein that recognizes the MPRs in late endosomes (TIP47), a pathway- specific SNARE complex for fusion of MPR-vesicles at the TGN, and two proteins that function in vesicle tethering at the Golgi (GCC185 and RhoBTB3). The goals of this application are (1) to define precisely, the distinct routes taken by cargoes that are transported from early endosomes back to the Golgi, with focus on MPRs in comparison with cholera toxin; (2) to carry out a genome-wide, automated siRNA screen for proteins needed for MPR recycling. The screen will make use of the fact that depletion of proteins needed for MPR recycling leads to dispersal of MPRs into peripherally localized cellular compartments. Computer software can detect this dispersal, permitting automated analysis of the effects of 22,000 siRNAs transfected into cultured cells. (3) Also proposed are experiments to further characterize two novel Rab9 effectors that are important for this trafficking pathway: RhoBTB3 and RUTBC1. In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of receptor trafficking in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders.

DESCRIPTION (provided by applicant): Niemann Pick type C (NPC) disease is a fatal pediatric disorder. The disease is due to mutations in either of two genes, NPC1, which encodes a 13 transmembrane domain sterol-binding protein, and NPC2, which encodes a soluble sterol-binding protein. Loss of either gene causes aberrant organelle trafficking and accumulation of free cholesterol within lysosomes. NPC patients suffer from hepatomegaly and progressive cognitive and locomotion losses, with massive Purkinje neuron (PN) death in the cerebellum. There is no effective treatment for NPC. Specific Aim 1: Determine why Purkinje neurons die in NPC disease and at what stages the disease can be arrested or reversed. We will provide a functional tagged- Npc1 protein to specific classes of cells, eg neurons, astrocyte, or liver cells, in the Npc1-/- background, to define how each cell type contributes to disease. Our transgenes are engineered with Tet-technology to allow cell-specific and temporal regulation of tagged Npc1 production. Specific Aim 2: Learn how NPC disease affects intracellular trafficking in Purkinje neurons. Loss of Npc1 causes striking intracellular trafficking defects in fibroblasts. To learn how PNs are affected, we will culture them and track the trafficking of NGF and other molecules labeled with quantum dots. We will use a portable two-photon microscope in combination with organelle dyes to characterize vesicular movements in living astrocytes and PNs of wild-type and Npc1-/- mice. In living brains of our newly engineered mice we will analyze movements of Npc1-positive organelles in PNs, other neurons, and astrocytes to determine what changes may cause cell death. Specific Aim 3: Determine whether inflammation protects from, or causes, NPC cell death. We will produce functional Npc1 in neurons, hepatocytes, and inflammatory cells, in otherwise Npc-/- mice, and see which of these is most effective in preventing inflammation. We will use mouse mutations that reduce inflammation in combination with Npc1-/-, and see whether PN survival and liver pathology are improved or worsened. We will ablate hepatic macrophages with chlodronate-filled liposomes and assess the role of macrophages in NPC liver damage. Specific Aim 4: Discover whether autophagy protects from, or causes, NPC cell death. We will cross NPC mice with mutants that cannot trigger autophagy: Toll like receptor-7 and beclin-1 deficient mice. Conversely we will test mice that have over-expressing Beclin1 in neurons and consequent have heightened autophagy, or enhance autophagy with Beclin1 virus infections or rapamycin treatments. Lysosome storage disorders like NPC encompass nearly 60 different conditions, most of which damage liver and/or brain function. Some, including NPC, have similarities to Alzheimer disease. We have used a novel approach to engineer mice that will allow us to learn how different cell types and processes contribute to disease. Learning the roles of inflammation and autophagy in NPC neurodegeneration has direct implications for therapeutic interventions that will arrest or reverse disease progression.

DESCRIPTION (provided by applicant): The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. We have shown that the protein, GCC185 is needed for tethering of MPRs at the Golgi. To investigate the mechanism of MPR vesicle tethering at the Golgi, we will analyze the structure of this purified Golgi tether using atomic force microscopy. This is important because transport vesicle tethering is a fundamental cell biological process that is poorly understood. We will test if the protein bends on the Golgi, in cells, and whether this bending is needed for its function. We will also determine the consequence of Rab GTPase binding on GCC185's conformation. This is important because GTPase binding is a common feature of Golgins and is likely to reflect the most physiological state of these proteins. We will next add fluorescently labeled, mannose 6-phosphate receptor-containing vesicles and monitor how these engage the GCC185 tether. Where do the vesicles bind? What models best explain how vesicles are tethered at the Golgi? Finally, we will study the very first step in retrograde transport: the loading of Rab9 onto late endosomes. For this, we will determine if Rab9A-specific DENND2 GEFs are part of a late endosomal Rab cascade. Understanding how DENND2 proteins first activate Rab9 will provide key information regarding establishment of the MPR retrograde trafficking pathway on late endosomes and formation of the late endocytic pathway. In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of receptor trafficking in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders.

Project Summary The long term goal of this research is to understand how cholesterol gains access to intracellular compartments for utilization, storage and cellular regulation, via two glycoproteins: NPC1 and LAMP proteins. NPC1 is needed for the egress of endocytosed, LDL-derived cholesterol from lysosomes into the cytoplasm. LAMP proteins constitute the major glycoproteins that line the limiting membranes of lysosomes. The goal of this application is to study how these proteins may cooperate to transport cholesterol out of lysosomes, using biochemical and cell biological approaches. Experiments are proposed to investigate further, the cholesterol-dependent interaction between these proteins, and how they, and their interactions, may influence downstream activation of lysosome biogenesis. These experiments will provide important information regarding how cells may sense and signal the availability of cholesterol in lysosomes, and export cholesterol from lysosomes. This work has important implications for a number of disease states including cardiovascular disease, cancer, and neurological disorders.