Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

doi:10.1083/jcb.139.5.1097

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© The Rockefeller University Press, 0021-9525/1997//1097 $5.00
The Journal of Cell Biology, Volume 139, Number 5, , 1997 1097-1108

Article

Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

Charles Barlowe

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

A cell-free vesicle fusion assay that reproduces a subreactionin transport of pro- ${alpha}$ -factor from the ER to the Golgi complexhas been used to fractionate yeast cytosol. Purified Sec18p,Uso1p, and LMA1 in the presence of ATP and GTP satisfies therequirement for cytosol in fusion of ER-derived vesicles withGolgi membranes. Although these purified factors are sufficientfor vesicle docking and fusion, overall ER to Golgi transportin yeast semi-intact cells depends on COPII proteins (componentsof a membrane coat that drive vesicle budding from the ER).Thus, membrane fusion is coupled to vesicle formation in ERto Golgi transport even in the presence of saturating levelsof purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derivedvesicles containing pro- ${alpha}$ -factor from docked vesicles and fromfused vesicles. Uso1p mediates vesicle docking and producesa dilution resistant intermediate. Sec18p and LMA1 are not requiredfor the docking phase, but are required for efficient fusionof ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPIIcoat) prevent vesicle fusion in a reversible manner, but donot interfere with vesicle docking. Ordering experiments usingthe dilution resistant intermediate and reversible Sec23p complexinhibition indicate Sec18p action is required before LMA1 function.

Abbreviations used in this paper: ARF, ADP-ribosylation factor; B88, buffer 88; GDI, GDP dissociation inhibitor; LMA1 and LMA2, low molecular weight activity 1 and 2; MSS, medium speed supernatant; NSF, N-ethylmaleimide–sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; YPD, yeast extract, peptone, dextrose.

I thank A. Haas, S. Sapperstein, G. Waters, and W. Wickner forgifts of plasmids, antibodies, and reagents. I am indebtedto Zuoyu Xu for his advice.

Address all correspondence to Charles Barlowe, Department ofBiochemistry, Dartmouth Medical School, Hanover, NH 03755. Tel.:(603) 650-6516. Fax: (603) 650-1353. E-mail: barlowe{at}dartmouth.edu

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Alvarez, C., Fujita, H., Hubbard, A., Sztul, E. (1999). ER to Golgi Transport: Requirement for P115 at a Pre-Golgi Vtc Stage. JCB 147: 1205-1222 [Abstract] [Full Text]
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Lustgarten, V., Gerst, J. E. (1999). Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis. Mol. Cell. Biol. 19: 4480-4494 [Abstract] [Full Text]
Spang, A., Schekman, R. (1998). Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro. JCB 143: 589-599 [Abstract] [Full Text]
Nelson, D. S., Alvarez, C., Gao, Y.-s., Garcia-Mata, R., Fialkowski, E., Sztul, E. (1998). The Membrane Transport Factor TAP/p115 Cycles between the Golgi and Earlier Secretory Compartments and Contains Distinct Domains Required for Its Localization and Function. JCB 143: 319-331 [Abstract] [Full Text]
VanRheenen, S. M., Cao, X., Lupashin, V. V., Barlowe, C., Gerard Waters, M. (1998). Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking. JCB 141: 1107-1119 [Abstract] [Full Text]
Jiang, Y., Al Scarpa, , Zhang, L., Stone, S., Feliciano, E., Ferro-Novick, S. (1998). A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport. Genetics 149: 833-841 [Abstract] [Full Text]
Klionsky, D. J. (1998). Nonclassical Protein Sorting to the Yeast Vacuole. J. Biol. Chem. 273: 10807-10810 [Full Text]
Orci, L., Perrelet, A., Rothman, J. E. (1998). Vesicles on strings: Morphological evidence for processive transport within the Golgi stack. Proc. Natl. Acad. Sci. USA 95: 2279-2283 [Abstract] [Full Text]
Hong, W (1998). Protein transport from the endoplasmic reticulum to the Golgi apparatus. J. Cell Sci. 111: 2831-2839 [Abstract]
Imoto, M, Tachibana, I, Urrutia, R (1998). Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p. J. Cell Sci. 111: 1341-1349 [Abstract]
Legesse-Miller, A., Sagiv, Y., Glozman, R., Elazar, Z. (2000). Aut7p, a Soluble Autophagic Factor, Participates in Multiple Membrane Trafficking Processes. J. Biol. Chem. 275: 32966-32973 [Abstract] [Full Text]
Orci, L., Ravazzola, M., Volchuk, A., Engel, T., Gmachl, M., Amherdt, M., Perrelet, A., Sollner, T. H., Rothman, J. E. (2000). Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional "percolating" COPI vesicles. Proc. Natl. Acad. Sci. USA 97: 10400-10405 [Abstract] [Full Text]