David D. Sabatini - US grants

We propose to study the process of formation of the carrier vesicles that mediate the transport of proteins from the trans Golgi network (TGN) to the cell surface of epithelial cells and to establish the mode of action of the specific proteins, lipids and cofactors that, in the cytosol and TGN membranes, play essential roles in this process. For these studies, we will use a cell-free system we have developed that recreates in vitro the generation of post Golgi vesicles from a purified Golgi fraction obtained from virus-infected MDCK cells. With this system, the formation of post Golgi vesicles can be effected in two sequential phases of: I) ArfGTP dependent coat assembly/bud formation and, 2) vesicle scission. The scission phase requires a PKC-like molecule in the TGN membrane, but not its phosphorylating activity, as well as several cytosolic proteins, including a membrane scission promoting activity (MSPA) present in a high molecular weight complex that contains an NEM-sensitive component, which we have tentatively identified as the phosphatidylinositol transfer protein (PITP). Employing a combined biochemical, genetic, and electron microscopic approach we will: l) Identify the coat and membrane components of TGN-derived vesicles by analysis of the coats of purified vesicles produced in the presence of GTPlambdaS, which prevents vesicle uncoating, and by assembling the vesicles with purified cytosolic subfractions containing coat subunits. 2) Purify and elucidate the regulation of a cytosolic NEM-sensitive membrane scission promoting factor (MSPA) that we have identified, which is required for vesicle generation and, when separated from other components, causes the uncontrolled vesiculation of uncoated TGN membranes. 3) Test a model in which vesicle scission is dependent on a regulatory mechanism that involves the action of a membrane-associated PKC-like molecule that, in concert with Arf-GTP, a cytosolic PITP that contributes PtdIns(4,5)P2, and possibly RhoA, leads to the activation of a phospholipase D (PLD), which, in TGN membranes, would be the final effector of vesicle scission. In these studies, we will identify the PKC-like molecule that we have implicated in vesicle generation and determine whether it interacts directly with and activates a Golgi associated PLD. We will also clarify the role of PITP and identify the phosphoinositides that it contributes to the scission process.

The long-term objective of this project is to understand the biogenetic mechanisms responsible for the subcellular distribution of specific proteins within membranes and organelles. The biosynthesis of several model proteins with diverse destinations, including the endoplasmic reticulum (e.g., cytochrome P-450 and its reductase), plasma membrane (e.g., erythrocyte band 3, the sodium-potassium ATPase and viral envelope glycoproteins), lysosomes (e.g., cathepsin D and b-glucuronidase) and mitochondria (e.g., cytochrome C and cytochrome oxidase) will be studied. For proteins which are synthesized in bound polysomes, features which serve as signals for their cotranslational insertion into the ER or which halt the vectorial discharge and lead to retention of polypeptides within the membranes will be identified. This will be accomplished using in vitro protein synthesis systems with added microsomal membranes and by analysis of the primary amino acid sequences derived from protein chemistry and cDNA sequencing studies. It is expected that the number and location of insertion and halt transfer signals in a membrane polypeptide will account for its transmembrane disposition. Sorting out processes which, following cotranslational insertion of a polypeptide into the ER, ensure its distribution within the cell will be investigated. Cytochemical studies on Golgi membranes will be carried out to relate the polarity of the Golgi complex to its role in sorting out mechanisms. The pathway of a polypeptide will be stutied using agents which perturb the transfer of products between organelles within cells and in vitro reconstruction systems for this transfer will be developed. The assignment of a specific role to polypeptide segments in the insertion, halt transfer or sorting processes will be aided by recombinant DNA procedures to construct modified and chimeric genes which will be introduced into cultural cells to follow the distribution of the products of their expression. Post-translational incorporation of polypeptides into mitochondria will also be investigated to study the mechanism of action of an addressing signal which was identified within cytochrome C and directs the uptake of this polypeptide into the organelle.

This is a proposal requesting funds for the purpose of a JeoL 1200 electron microscope to replace an obsolescent Philips 301. A variety of studies, which demand high quality electron microscopy as a prerequisite for their success, will benefit from that acquisition of the new microscope. The proposed projects will investigate: a) the posttranslational sorting of proteins out of the endoplasmic reticulum and to cellular organelles, (b) the polarized delivery of viral glycoproteins to the plasma membranes of epithelial cells, (c) aspects of the process of ribosome attachment to the endoplasmic reticulum, (d) the distribution and functional expression of central nervous system myelin (e) the differentiation and modulation of steroid secreting cells, (f) the role of the myelin associated glycoprotein in myelination of nerves, (g) the role of a small molecular weight metabolic in cystic fibrosis, (h) the synthesis and secretion of cellular growth regulatory factors, (i) the inhibition of the complement cascade, (j) the characterization of plasmodial antigens, (k) the expression of keratins and membrane proteins during differentiation of epithelia, (1) the uptake and metabolism of vitamin E. The above projects are largely being undertaken by a number of investigators in the Department of Cell Biology, who have over the years harmoniously shared major pieces of equipment and other resources within the department. In addition, several other users from outside the department will share the facility. The proposed microscope is especially suited as a shared instrument.

The overall goal of this proposal is to elucidate the sorting mechanisms that serve to establish the distinct protein compositions of the two plasma membrane domains of polarized epithelial cells. We wish to determine: i) how newly synthesized membrane proteins emerging from the trans Golgi network (TGN) are incorporated into specific vesicles, and, ii) how these vesicles are directed to one aspect of the cell surface. We propose to test a model in which certain proteins (Class I) sort by themselves by interaction in the TGN with adaptor molecules that recognize their cytoplasmic segments and lead to the incorporation of the proteins into vesicles directed to the plasma membrane. Other proteins, such as the HA of influenza and G of VSV, would be recognized by sorting receptors, which are Class I molecules that interact with the luminal domains of the proteins to be sorted and transport them to the cell surface. Polarized cell monolayers, perforated in one or the other surface, and a cell-free system, will be employed to identify, and ultimately purify, cellular components, including adaptor proteins and GTP-binding proteins and their cognate docking proteins, that participate in the formation and targeting of the transport vesicles. Complexes between the viral glycoproteins and their putative sorting receptors and the corresponding adaptors will be searched for in total cell extracts and subcellular fractions, including purified post Golgi vesicles. To investigate the role in sorting of the cytoplasmic segments of proteins that sort by themselves, peptides corresponding to the cytoplasmic tails of various receptors will be used in permeabilized cells and in the cell-free system, in attempts to specifically inhibit transport to one or the other aspect of the cell surface. The specific association of the cytoplasmic segments with adaptor-like molecules will be directly investigated by cross-linking and label-transfer techniques and by attempts to purify the adaptors using the cytoplasmic segments as affinity ligands. The perforated cell system will be used to examine the requirements for the formation of endocytic vesicles from each surface and for the transport of these vesicles within the cell that leads to their fusion with endosomes. The role of specific cytoskeletal elements, such as actin and fodrin, in endocytosis at each surface will be examined.

This application requests funds for the purchasing of the components needed to establish an integrated ultracryomicrotome system for the preparation and sectioning of frozen or plastic-embedded biological specimens to be examined by immunoelectron microscopy. This technologically advanced system allows the preservation of fine structural details, while avoiding or minimizing the use of the chemical fixatives, organic solvents or resin polymerization conditions that cause extensive protein denaturation and loss of antigenic epitopes. This make it possible to localize by immunolabeling proteins of interest at the level of resolution attained by electron microscopy. The six investigators that participate in this application are all interested in localizing specific gene products within cultured cells or animal tissues. They work in the areas of protein trafficking (Sabatini/Adesnik, Philips), cell-cell interactions (Cowin, Salzer) and pathogen-host relationships (Zychlinsky, Nussenzweig/Frevert). The system we plan to establish will be located in the electron microscopy suite of the Cell Biology Department, isolated from airdrafts and sudden temperature changes, and will be operated by a skilled and experienced technician, supervised by an accomplished electron microscopist with 15 years experience in immunolabelling techniques. The system we request consists of a Leica UCT ultramicrotome, the Leica FC-S low temperature sectioning attachment, and the Leica equipment necessary for cryopreparation by the rapid freezing technique, and for embedding by cryosubstitution. Funds are also requested to acquire cryotools, an ionizer that minimizes section chattering, and an automatic section stainer.