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Intracellular Transport of Proteins
Summary: Randy Schekman's research is focused on the process of membrane assembly, vesicular transport, and membrane fusion among organelles of the secretory pathway.
Our research is devoted to a molecular description of the process of membrane assembly and vesicular traffic in eukaryotic cells. Basic principles that emerged from these studies in yeast are now being applied to studies of genetic diseases of protein transport.
A combination of genetic and cytologic evaluation of the secretion (sec) mutants has allowed a description of the secretory pathway in Saccharomyces cerevisiae. 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.
Vesicle Transport Early in the Secretory Pathway
We have developed biochemical assays that measure the early events of polypeptide translocation into the endoplasmic reticulum (ER) and of vesicle-mediated protein transport from the ER to the Golgi apparatus. Transport of secretory and membrane cargo proteins 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; 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 (Sar1p, Sec23/24p, and Sec13/31p) 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 membrane shape change that accompanies vesicle budding. Liposomes formulated with phospholipids representative of a yeast ER membrane fraction bind the COPII proteins 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.
Vesicle Traffic Late in the Secretory Pathway
Little is known about the mechanism of sorting and packaging of secretory proteins that transit from the Golgi complex to the cell surface. Although some proteins use clathrin to traverse the endosome en route to the plasma membrane, others do not, and, until now, the general view has been that the direct path out of the trans-Golgi network (TGN) may involve tubular carriers formed without the intervention of coat proteins. To examine this limb of the secretory pathway, we have been supported by a grant from the National Institutes of Health to study the transport of a cell wall biosynthetic enzyme, chitin synthase III (Chs3p), from the TGN/endosome membrane to the plasma membrane of the mother/bud junction in yeast.
Genetic and biochemical analyses have now shown that Chs3p and a small set of other cell surface proteins are conveyed from the TGN by a novel coat protein complex we call exomer. The exomer consists of five subunits: Chs5p, Chs6p, and nonstoichiometric levels of three paralogs of Chs6p. Exomer is a large, ~1-MDa complex that binds membranes in the presence of a GTP-activated form of Arf1p, a small GTPase implicated in other coat protein assembly events. Like COPII, exomer recognizes a sorting signal that in this case specifies TGN to cell surface transport. Unlike other coats, however, exomer is responsible for the traffic of a nonessential set of surface proteins. The vesicle or tubule morphogenesis by exomer and the discovery of other coats involved in the traffic of other cell surface molecules are subjects of investigation in our lab.
Traffic in Human Genetic Diseases
Sec23 has been implicated in a rare craniofacial disorder (CLSD) that causes various skeletal defects. The CLSD mutation maps to a residue on the surface of Sec23 that is away from contact with the membrane. Although the mutation, F382L, produces a conservative substitution, the effect of the allele on primary fibroblasts cultured from patients is profound, with a substantial distortion of the ER reminiscent of the original and lethal sec23-1 of yeast. Humans have two paralogs of Sec23, but cultured fibroblasts and calvarial osteoblasts, which may be responsible for the skeletal effects in CLSD patients, have only one copy, Sec23A. We have established a vesicle-budding reaction reconstituted with mammalian COPII proteins and ER membranes to explore CLSD and other unique features of human membrane protein traffic. Using pure recombinant proteins, we found that the CLSD mutation interferes with the binding and assembly of a scaffold complex, Sec13/31, which normally builds upon the inner coat of Sar1p and Sec23/24p.
Membrane proteins implicated in familial forms of Alzheimer's disease (FAD) are substrates for vesicular trafficking, and defects associated with protein transport may play a role in the pathology of AD. Presenilin 1 (PS1) is an essential subunit of an enzyme, γ-secretase, that serves important roles in the maturation of proteins involved in signaling and development. However, mutant forms of PS1 cause unscheduled processing of amyloid precursor protein (APP) to generate an amyloidogenic peptide that accumulates in brain neuritic plaques that are characteristic of AD. Surprisingly, the PS1 mutations that potentiate the action of γ-secretase on APP are spread throughout the molecule, including in domains exposed to the cytoplasm, to the lumen of the ER or extracellular space, and within the membrane bilayer. These alleles may produce forms of PS1 that fold improperly and retard the traffic of γ-secretase from the ER. Such impaired traffic could influence the transport and proteolytic processing of APP.
In work initiated with support from private donors, including the Adler Foundation, we used mammalian COPII proteins to explore the packaging of APP and PS1 in lysates of fibroblast and neuronally derived cell lines. PS1 is enriched in the ER and Golgi membrane in precursor and autoproteolytically processed forms. Precursor forms of PS1 and the γ-secretase complex are packaged along with APP into COPII vesicles, although the requirements for packaging are distinct. Cell lines derived from mouse PS knockout strains are proficient in the packaging of APP. However, certain FAD mutant forms of PS1 retard the incorporation of mutant PS1 and apparently intact γ-secretase complex. Retention of the γ-secretase in the ER could lead to aberrant processing of APP and to the production and secretion of the amyloidogenic peptide Aβ42. Reconstitution of the formation of the amyloid peptide in the context of a cell-free transport reaction may illuminate the earliest stages in the pathology of AD.
Last updated: May 1, 2008
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