Biography
Research interests:
Research in our laboratory is devoted to a molecular description of the process of polypeptide translocation from the cytosol into the endoplasmic reticulum (ER) and of vesicular transport and membrane fusion among organelles of the secretory pathway.
Genetic and Biochemical Dissection of the Secretory Process
In a genetic approach to the study of eukaryotic protein transport, we isolated a series of secretory (sec) mutants in the yeast Saccharomyces cerevisiae that are temperature sensitive for cell surface growth, division, and secretion. Most of these mutants accumulate secretory proteins in an intracellular pool that can be released when cells are returned to a permissive temperature. More than 30 gene products have been implicated in the process of delivering membrane and secretory proteins to the cell surface.
A combination of genetic and cytologic evaluation of the sec mutants has allowed a description of the secretory pathway. Protein transport in yeast appears to be mediated by the same organelles and proteins that operate in mammalian cells. Molecular cloning analysis of SEC genes revealed striking structural and functional homology with corresponding mammalian genes.
We have developed biochemical assays that measure the early events of polypeptide translocation into the ER and of vesicle-mediated protein transport from the ER to the Golgi apparatus. The cell-free reactions represent physiologically meaningful processes. Extracts prepared from mutant cells reproduce the temperature-sensitive defects observed in vivo. In favorable circumstances, the mutant defects are repaired by addition of a protein fraction obtained from wild-type yeast. Such restoration of transport activity has been used to purify functional forms of SEC gene products.
Vesicle Transport Early in the Secretory Pathway
Transport of yeast a-factor precursor is mediated by diffusible vesicles. The formation of these vesicles in vitro depends on the Sec proteins that were predicted to be involved from genetic and morphological inspection of sec mutant cells. Isolated transport vesicles contain membrane and internal proteins that are targeted to other compartments in the cell, but they are nearly devoid of proteins that are located in the ER. Thus the budding mechanism somehow distinguishes transported from ER-resident proteins. This sorting and budding process is highly evolutionarily conserved, because mammalian equivalents of the yeast Sec proteins have been isolated and are known to operate in the same location within the cell.
Vesicles formed in the transport reaction have an electron-dense, 10-nm coat structure that consists of the Sec proteins required in budding. This coat (COPII) resembles another coat complex (COPI) that creates transport vesicles within the Golgi apparatus. Our working model is that the Sec protein subunits of the COPII coat bind to the ER membrane and recruit cargo molecules into a cluster that then dimples the membrane to form a bud. A direct interaction between one of the COPII subunits, Sec24p, and membrane proteins is implicated in the capture of cargo proteins. This capture results in the concentrative sorting of membrane and secretory proteins, the latter being selected by an indirect interaction mediated by various membrane receptor proteins that link the coat to soluble cargo proteins. Fission of the bud from the membrane separates transported from resident proteins.
In addition to a role in cargo selection, the COPII coat is responsible for the change in membrane shape that accompanies vesicle budding. Liposomes formulated with phospholipids representative of a yeast ER membrane fraction bind the COPII protein in the same sequence of events and with the same nucleotide dependence as observed with native ER membrane. Furthermore, COPII buds and vesicles form on the surface of the liposome and capture solute from the interior of the liposome. Other coat protein complexes (clathrin and COPI) display similar budding activity on synthetic membrane liposomes.
Individual steps in the assembly of the coat on liposomes may be monitored by light scattering. This assay allows sufficient time resolution to detect transient events such as those accompanying nucleotide exchange and GTP hydrolysis mediated by a key regulatory molecule of COPII, Sar1p.
The mechanism by which COPII recruits membrane proteins in the ER may have a direct bearing on the transport and processing of mammalian proteins implicated in familial forms of Alzheimer's disease (FAD). Three loci, encoding amyloid precursor protein (APP), presenilin I (PSI), and PSII, have been identified in families genetically predisposed to AD. Aberrant proteolytic processing of APP to create the amyloidogenic peptide Ab 1?42 is strongly correlated to brain lesions and AD. This misprocessing occurs either in the ER or shortly after APP is transported out of the ER. PSI and -II are ER membrane proteins that may carry APP out of the ER and expose it to normal or unscheduled proteolysis. PSI, PSII, and APP may expose signals for the packaging of a protein complex into COPII vesicles. We are using the observation that yeast COPII proteins sort membrane proteins and bud transport vesicles from mammalian ER membranes to address these issues. Yeast COPII proteins and GTP mixed with ER membranes from Chinese hamster ovary (CHO) cells form hybrid COPII vesicles that contain APP and a precursor form of PSI but not resident ER proteins such as ribophorin. Wild-type and FAD forms of PSI are being investigated in this hybrid reaction.
Transport of Membrane Proteins to the Division Septum
Most new cell surface proteins are assembled in the bud portion of a growing yeast cell. However, some membrane proteins define a ring that separates the bud and mother portions of a cell. We have focused on the enzymes that make chitin, a carbohydrate polymer in the cell wall that grows inward from a ring to form a disk that separates daughter cells at the end of the cell division cycle. Chitin synthases (CSI, -II, and -III) operate at different stages in the cell cycle to form the ring (CSIII) or the division septum (CSII). All three enzymes traverse the secretory pathway en route to the cell surface; however, CSII and -III then end up tightly organized at the locus where chitin is deposited.
Surprisingly, the cell employs different mechanisms to organize the temporal and spatial deployment of CSII and -III. CSII synthesis is regulated to ensure its availability is limited to the peak period of division septum formation. After cell separation, CSII is endocytically internalized and delivered to the vacuole, where it is degraded. In contrast, CSIII synthesis and degradation are not regulated. Instead, CSIII cycles between a late compartment of the Golgi apparatus and the cell surface. We propose that the late-Golgi compartment is a holding station from which CSIII may be mobilized back to the mother-daughter cell junction at the beginning of the cell cycle. Our major task now is to understand the mechanism and regulation of this novel transport reaction.
We have developed genetic selections to identify genes required for the trafficking of CSIII from the trans-Golgi to the cell surface. We have also established a permeabilized cell system, similar to that employed in our discovery of COPII, that reproduces the formation of transport vesicles from the trans-Golgi membrane. The combination of genetics and biochemistry should allow us to describe the molecular mechanism of protein sorting late in the secretory pathway.
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