Howard Hughes Medical Institute Investigator and Professor of Cell Biology, Development and Physiology*
*And Affiliate, Division of Biochemistry and Molecular Biology
Our research is devoted to a molecular description of the process of membrane assembly and vesicular traffic in eukaryotic cells. Basic principles and approaches that we developed from studies on a simple eukaryote, Saccharomyces cerevisiae, are now being applied to investigate the mechanisms of intracellular vesicular traffic and the biogenesis of extracellular vesicles (exosomes) in cultured human cells.
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. Vesicles formed in the transport reaction have an electron-dense, 10-nm coat structure that consists of the Sec proteins (the GTP binding protein 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 (1).
Traffic of large and unusually shaped cargo complexes such as procollagen (PC1, 300 nm rod-shaped oligomeric protein) and lipoprotein particles (carrying apolipoprotein B and cholesterol) display special requirements to form larger-than-normal COPII coat complexes. Work in collaboration with the Rape lab led to the discovery of a role for ubiquitylation of the Sec31 subunit of the COPII coat in the capture of procollagen into large COPII vesicles (2). These large, PC1-containing vesicles have been observed in cultured human cells, and in a cell-free COPII vesicle budding reaction (3). The capture of PC1 into larger-than-normal COPII vesicles may be directly influenced by the co-packaging of the Sar1 guanine exchange factor (GEF), Sec12, bound to the ER membrane receptor for PC1, TANGO1 (4). In addition, lipoprotein sorting from the ER appears to require the intervention of a fatty acid binding protein, FABP5, as well as the COPII machinery for particle packaging into transport vesicles (5).
We have also reported a connection between the COPII coat and the formation of the autophagosome, an organelle responsible for the capture of protein aggregates and organelles that are delivered to and degraded in the lysosome. By following the covalent attachment of a lipid group, phosphatidylethanolamine, to the C-terminus of a cytoplasmic protein, LC3, we found that an organelle that mediates vesicular flow between the ER and the Golgi apparatus, the ERGIC, is the membrane that gives rise to an autophagosome precursor organelle (6). Further, we found that the ERGIC forms novel COPII vesicles that build the autophagosome membrane (7).
Although the standard secretory pathway accommodates the majority of cargo proteins exported by eukaryotic cells, novel processes invoking other membranes have evolved to accommodate special regulatory needs. We have studied several small, soluble cytoplasmic proteins, interleukin-1β (IL-1β), fatty acid binding protein 4 (FABP4) and alpha synuclein, each of which employs an alternative route that bypasses the normal secretory pathway. We reported that proteolytically processed IL-1β translocates through the outer membrane of the autophagosome envelope to reside in the luminal space between the two membranes (8). Extracellular secretion of mature IL-1β would then follow if the autophagosome fused directly to the plasma membrane or first with a lysosomal intermediate organelle. The nature of the translocation channel in the autophagosome outer membrane is now being pursued in the new independent laboratory of the postdoctoral couple who made this discovery in my lab.
Export of FABP4 appears to invoke the lysosome although not the autophagosome for unconventional secretion from adipocytes (9). Here also, the translocation channel responsible for the export of FABP4 remains unknown.
Current effort on alpha synuclein, a protein genetically implicated in Parkinson’s Disease (PD), is focused on the means by which this soluble, relatively unstructured protein may be translocated across a membrane without a typical signal peptide. Alpha synuclein is a major constituent of Lewy Body particles that appear in the dopaminergic neurons in patients who have died of PD. One question is how synuclein may progress out of dopaminergic neurons, in a midbrain tissue called the substantia nigra, into other areas of the brain as may happen in the spread of dementia in some patients with PD.
An entirely distinct form of unconventional secretion invokes the capture of cytoplasmic proteins and small RNA molecules into vesicles exported into the extracellular medium in cultured cells or the extracellular fluids in metazoan organisms. Extracellular vesicles (EVs) form in two distinct pathways. Microvesicles bud directly from the cell surface into the extracellular medium whereas a distinct subset of EVs are formed by membrane invagination into the interior of a late endosome to create a multivesicular body (MVB) which by fusion at the cell surface releases a bolus of EVs called exosomes. Much interest has developed around the evidence that EVs may serve as a means of intercellular communication in which proteins and small RNAs are delivered by fusion or uptake into target cells to mediate control of metabolism or gene expression. Of particular interest is the evidence that the microRNA content of EVs in the blood may change as a result of metastatic cancer and that EVs produced abundantly by tumor cells may communicate with other tissues in the body to create a premetastatic niche into which primary tumor cells may migrate to form a secondary metastatic event (10).
We have devised fractionation procedures to isolate distinct populations of EVs from normal and tumor cells grown in culture. Two vesicle types produced by a human breast cancer cell line are resolved on a buoyant density gradient and each contains a largely non-overlapping set of miRNAs (11). One vesicle type contains several highly selected mature miRNAs which we estimate are ~1000X enriched over their content in the cytoplasm of the tumor cell. A human embryonic cell line produces exosomes that also have several highly selected miRNAs but these differ from those enclosed within the selective pool of vesicles produced by the breast cancer cells (12, 13). These results suggest a cell type-specific high-fidelity sorting reaction to secrete only a subset of miRNAs from cells. This sorting may serve the purpose of selective disposal of certain miRNAs or the targeted delivery of selected miRNAs for control of gene expression in cells that take up and functionally incorporate the exosome content.
In order to understand the molecular mechanism of RNA sorting in the selection of certain miRNAs for packaging into exosomal vesicles, we have devised a cell-free reaction that recapitulates this process (11, 12). Chemically synthetic mature miRNAs are mixed with ATP membranes and cytosolic proteins isolated from broken cells, and after a period of incubation at 30C, the fraction of miRNA sequestered within membranes and thus protected against degradation by exogenous RNase is quantified by qt-PCR. We observe RNA sequence-selective uptake of certain miRNAs into vesicles in a reaction that depends upon the presence of membranes and cytosolic proteins and is stimulated 2-fold by ATP. Using biotinylated derivatives of two different sorted miRNAs, we have obtained evidence for roles of two RNA binding proteins, Ybx1 and the La antigen. Further cellular and biochemical investigations are underway to uncover the molecular basis of this high-fidelity sorting reaction.
COPII and the regulation of protein sorting in mammals. Zanetti G, Pahuja KB, Studer S, Shim S, Schekman R. Nat Cell Biol. 2011 Dec 22;14(1):20-8. doi: 10.1038/ncb2390. Review. Erratum in: Nat Cell Biol. 2012 Feb;14(2):221.
Ubiquitin-dependent regulation of COPII coat size and function. Jin L, Pahuja KB, Wickliffe KE, Gorur A, Baumgärtel C, Schekman R, Rape M. Nature. 2012 Feb 22;482(7386):495-500. doi: 10.1038/nature10822.
COPII-coated membranes function as transport carriers of intracellular procollagen I. Gorur A, Yuan L, Kenny SJ, Baba S, Xu K, Schekman R. J Cell Biol. 2017 Jun 5;216(6):1745-1759. doi: 10.1083/jcb.201702135. Epub 2017 Apr 20.
TANGO1 and SEC12 are copackaged with procollagen I to facilitate the generation of large COPII carriers. Yuan L, Kenny SJ, Hemmati J, Xu K, Schekman R.Proc Natl Acad Sci U S A. 2018 Dec 26;115(52):E12255-E12264. doi: 10.1073/pnas.1814810115. Epub 2018 Dec 13.
Fatty-acid binding protein 5 modulates the SAR1 GTPase cycle and enhances budding of large COPII cargoes. Melville D, Gorur A, Schekman R. Mol Biol Cell. 2019 Feb 1;30(3):387-399. doi: 10.1091/mbc.E18-09-0548. Epub 2018 Nov 28.
The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. Ge L, Melville D, Zhang M, Schekman R. Elife. 2013 Aug 6;2:e00947. doi: 10.7554/eLife.00947.
Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. Ge L, Zhang M, Schekman R. Elife. 2014 Nov 28;3:e04135.
Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion. Zhang M, Kenny SJ, Ge L, Xu K, Schekman R. Elife. 2015 Nov 2;4. pii: e11205.
Unconventional secretion of FABP4 by endosomes and secretory lysosomes.
Villeneuve J, Bassaganyas L, Lepreux S, Chiritoiu M, Costet P, Ripoche J, Malhotra V, Schekman R. J Cell Biol. 2018 Feb 5;217(2):649-665. doi: 10.1083/jcb.201705047. Epub 2017 Dec 6.
Extracellular Vesicles and Cancer: Caveat Lector Matthew J. Shurtleff, Morayma M. Temoche-Diaz, Randy Schekman. Annual Review of Cancer Biology 2018 2:1, 395-411.
Distinct mechanisms of microRNA sorting into cancer-derived extracellular vesicle subtypes. Temoche-Diaz, M., Shurtleff, M.J., Nottingham, R., Yao, J., Fadadu, R. P., Lambowitz, A.and Schekman, R. (preprint posted on bioRxiv: http://biorxiv.org/cgi/content/short/612069v1).
Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction. Shurtleff MJ, Temoche-Diaz MM, Karfilis KV, Ri S, Schekman R. Elife. 2016 Aug 25;5. pii: e19276. doi: 10.7554/eLife.19276.
Broad role for YBX1 in defining the small noncoding RNA composition of exosomes.
Shurtleff MJ, Yao J, Qin Y, Nottingham RM, Temoche-Diaz MM, Schekman R, Lambowitz AM.Proc Natl Acad Sci U S A. 2017 Oct 24;114(43):E8987-E8995. doi: 10.1073/pnas.1712108114. Epub 2017 Oct 10.
Last Updated 2019-04-17