Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

doi:10.1083/jcb.139.5.1097

This Article

	Full Text
	Full Text (PDF, 332K)
	PPT slides of all figures
	Alert me when this article is cited
	Citation Map

Services

	Email this article
	Similar articles in this journal
	Similar articles in PubMed
	Alert me to new content in the JCB
	Download to citation manager

Citing Articles

	Citing Articles via HighWire
	Citing Articles via CrossRef
	Citing Articles via Google Scholar

Google Scholar

	Articles by Barlowe, C.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by Barlowe, C.


Social Bookmarking

	What's this?

© 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

CiteULike

Complore

Connotea

Del.icio.us Add to Digg

Digg

Facebook

Technorati

Twitter What's this?

This article has been cited by other articles:

Lorente-Rodriguez, A., Heidtman, M., Barlowe, C. (2009). Multicopy suppressor analysis of thermosensitive YIP1 alleles implicates GOT1 in transport from the ER. J. Cell Sci. 122: 1540-1550 [Abstract] [Full Text]
Kamena, F., Diefenbacher, M., Kilchert, C., Schwarz, H., Spang, A. (2008). Ypt1p is essential for retrograde Golgi-ER transport and for Golgi maintenance in S. cerevisiae. J. Cell Sci. 121: 1293-1302 [Abstract] [Full Text]
Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J.-C., McCaffery, J. M., Barlowe, C., Lindquist, S. (2008). The Parkinson's disease protein {alpha}-synuclein disrupts cellular Rab homeostasis. Proc. Natl. Acad. Sci. USA 105: 145-150 [Abstract] [Full Text]
Behnia, R., Barr, F. A., Flanagan, J. J., Barlowe, C., Munro, S. (2007). The yeast orthologue of GRASP65 forms a complex with a coiled-coil protein that contributes to ER to Golgi traffic. JCB 176: 255-261 [Abstract] [Full Text]
Li, L., Shimada, T., Takahashi, H., Ueda, H., Fukao, Y., Kondo, M., Nishimura, M., Hara-Nishimura, I. (2006). MAIGO2 Is Involved in Exit of Seed Storage Proteins from the Endoplasmic Reticulum in Arabidopsis thaliana. Plant Cell 18: 3535-3547 [Abstract] [Full Text]
Welsh, L. M., Tong, A. H. Y., Boone, C., Jensen, O. N., Otte, S. (2006). Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex. J. Cell Sci. 119: 4730-4740 [Abstract] [Full Text]
Yu, S., Satoh, A., Pypaert, M., Mullen, K., Hay, J. C., Ferro-Novick, S. (2006). mBet3p is required for homotypic COPII vesicle tethering in mammalian cells. JCB 174: 359-368 [Abstract] [Full Text]
Brandon, E., Szul, T., Alvarez, C., Grabski, R., Benjamin, R., Kawai, R., Sztul, E. (2006). On and Off Membrane Dynamics of the Endoplasmic Reticulum-Golgi Tethering Factor p115 In Vivo. Mol. Biol. Cell 17: 2996-3008 [Abstract] [Full Text]
Flanagan, J. J., Barlowe, C. (2006). Cysteine-Disulfide Cross-linking to Monitor SNARE Complex Assembly during Endoplasmic Reticulum-Golgi Transport. J. Biol. Chem. 281: 2281-2288 [Abstract] [Full Text]
Sztul, E., Lupashin, V. (2006). Role of tethering factors in secretory membrane traffic. Am. J. Physiol. Cell Physiol. 290: C11-C26 [Abstract] [Full Text]
Ungermann, C., Langosch, D. (2005). Functions of SNAREs in intracellular membrane fusion and lipid bilayer mixing. J. Cell Sci. 118: 3819-3828 [Abstract] [Full Text]
Palmer, K. J., Konkel, J. E., Stephens, D. J. (2005). PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell Sci. 118: 3839-3847 [Abstract] [Full Text]
Kraynack, B. A., Chan, A., Rosenthal, E., Essid, M., Umansky, B., Waters, M. G., Schmitt, H. D. (2005). Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast. Mol. Biol. Cell 16: 3963-3977 [Abstract] [Full Text]
Miller, E. A., Liu, Y., Barlowe, C., Schekman, R. (2005). ER-Golgi Transport Defects Are Associated with Mutations in the Sed5p-binding Domain of the COPII Coat Subunit, Sec24p. Mol. Biol. Cell 16: 3719-3726 [Abstract] [Full Text]
Beard, M., Satoh, A., Shorter, J., Warren, G. (2005). A Cryptic Rab1-binding Site in the p115 Tethering Protein. J. Biol. Chem. 280: 25840-25848 [Abstract] [Full Text]
Heidtman, M., Chen, C. Z., Collins, R. N., Barlowe, C. (2005). Yos1p Is a Novel Subunit of the Yip1p-Yif1p Complex and Is Required for Transport between the Endoplasmic Reticulum and the Golgi Complex. Mol. Biol. Cell 16: 1673-1683 [Abstract] [Full Text]
Ballew, N., Liu, Y., Barlowe, C. (2005). A Rab Requirement Is Not Bypassed in SLY1-20 Suppression. Mol. Biol. Cell 16: 1839-1849 [Abstract] [Full Text]
Chen, C. Z., Calero, M., DeRegis, C. J., Heidtman, M., Barlowe, C., Collins, R. N. (2004). Genetic Analysis of Yeast Yip1p Function Reveals a Requirement for Golgi-Localized Rab Proteins and Rab-Guanine Nucleotide Dissociation Inhibitor. Genetics 168: 1827-1841 [Abstract] [Full Text]
Liu, Y., Flanagan, J. J., Barlowe, C. (2004). Sec22p Export from the Endoplasmic Reticulum Is Independent of SNARE Pairing. J. Biol. Chem. 279: 27225-27232 [Abstract] [Full Text]
Kamena, F., Spang, A. (2004). Tip20p Prohibits Back-Fusion of COPII Vesicles with the Endoplasmic Reticulum. Science 304: 286-289 [Abstract] [Full Text]
Heidtman, M., Chen, C. Z., Collins, R. N., Barlowe, C. (2003). A role for Yip1p in COPII vesicle biogenesis. JCB 163: 57-69 [Abstract] [Full Text]
Brands, A., Ho, T.-h. D. (2002). Function of a Plant Stress-Induced Gene, HVA22. Synthetic Enhancement Screen with Its Yeast Homolog Reveals Its Role in Vesicular Traffic. Plant Physiol. 130: 1121-1131 [Abstract] [Full Text]
Kosodo, Y., Noda, Y., Adachi, H., Yoda, K. (2002). Binding of Sly1 to Sed5 enhances formation of the yeast early Golgi SNARE complex. J. Cell Sci. 115: 3683-3691 [Abstract] [Full Text]
Liu, Y., Barlowe, C. (2002). Analysis of Sec22p in Endoplasmic Reticulum/Golgi Transport Reveals Cellular Redundancy in SNARE Protein Function. Mol. Biol. Cell 13: 3314-3324 [Abstract] [Full Text]
Wang, W., Ferro-Novick, S. (2002). A Ypt32p Exchange Factor Is a Putative Effector of Ypt1p. Mol. Biol. Cell 13: 3336-3343 [Abstract] [Full Text]
Scales, S. J., Hesser, B. A., Masuda, E. S., Scheller, R. H. (2002). Amisyn, a Novel Syntaxin-binding Protein That May Regulate SNARE Complex Assembly. J. Biol. Chem. 277: 28271-28279 [Abstract] [Full Text]
Pullikuth, A. K., Gill, S. S. (2002). In vivo membrane trafficking role for an insect N-ethylmaleimide-sensitive factor which is developmentally regulated in endocrine cells. J. Exp. Biol. 205: 911-926 [Abstract] [Full Text]
Powers, J., Barlowe, C. (2002). Erv14p Directs a Transmembrane Secretory Protein into COPII-coated Transport Vesicles. Mol. Biol. Cell 13: 880-891 [Abstract] [Full Text]
Whyte, J. R. C., Munro, S. (2002). Vesicle tethering complexes in membrane traffic. J. Cell Sci. 115: 2627-2637 [Abstract] [Full Text]
Belden, W. J., Barlowe, C. (2001). Role of Erv29p in Collecting Soluble Secretory Proteins into ER-Derived Transport Vesicles. Science 294: 1528-1531 [Abstract] [Full Text]
Spang, A., Herrmann, J. M., Hamamoto, S., Schekman, R. (2001). The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum. Mol. Biol. Cell 12: 1035-1045 [Abstract] [Full Text]
Otte, S., Belden, W. J., Heidtman, M., Liu, J., Jensen, O. N., Barlowe, C. (2001). Erv41p and Erv46p: New Components of Copii Vesicles Involved in Transport between the ER and Golgi Complex. JCB 152: 503-518 [Abstract] [Full Text]
Wang, W., Sacher, M., Ferro-Novick, S. (2000). Trapp Stimulates Guanine Nucleotide Exchange on Ypt1p. JCB 151: 289-296 [Abstract] [Full Text]
Dirac-Svejstrup, A. B., Shorter, J., Waters, M. G., Warren, G. (2000). Phosphorylation of the Vesicle-Tethering Protein P115 by a Casein Kinase II-Like Enzyme Is Required for Golgi Reassembly from Isolated Mitotic Fragments. JCB 150: 475-488 [Abstract] [Full Text]
Allan, B. B., Moyer, B. D., Balch, W. E. (2000). Rab1 Recruitment of p115 into a cis-SNARE Complex: Programming Budding COPII Vesicles for Fusion. Science 289: 444-448 [Abstract] [Full Text]
Weber, T., Parlati, F., McNew, J. A., Johnston, R. J., Westermann, B., Sollner, T. H., Rothman, J. E. (2000). Snarepins Are Functionally Resistant to Disruption by Nsf and {alpha}SNAP. JCB 149: 1063-1072 [Abstract] [Full Text]
Cao, X., Barlowe, C. (2000). Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes. JCB 149: 55-66 [Abstract] [Full Text]
Steel, G. J., Harley, C., Boyd, A., Morgan, A. (2000). A Screen for Dominant Negative Mutants of SEC18 Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis. Mol. Biol. Cell 11: 1345-1356 [Abstract] [Full Text]
Price, A., Wickner, W., Ungermann, C. (2000). Proteins Needed for Vesicle Budding from the Golgi Complex Are Also Required for the Docking Step of Homotypic Vacuole Fusion. JCB 148: 1223-1230 [Abstract] [Full Text]
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]
VanRheenen, S. M., Cao, X., Sapperstein, S. K., Chiang, E. C., Lupashin, V. V., Barlowe, C., Waters, M. G. (1999). Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p. JCB 147: 729-742 [Abstract] [Full Text]
Kim, D.-W., Sacher, M., Scarpa, A., Quinn, A. M., Ferro-Novick, S. (1999). High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking. Mol. Biol. Cell 10: 3317-3329 [Abstract] [Full Text]
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]