The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins.
Open Access
- 1 November 1993
- journal article
- research article
- Published by Springer Nature in The EMBO Journal
- Vol. 12 (11) , 4073-4082
- https://doi.org/10.1002/j.1460-2075.1993.tb06091.x
Abstract
SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a SEC13‐dihydrofolate reductase (mouse) fusion gene (SEC13:DHFR) was constructed that complements both sec13 temperature sensitive and null mutations. Methotrexate‐agarose affinity chromatography facilitated the purification of two forms of the Sec13‐dhfrp fusion protein: a monomeric form and a high molecular weight complex. The complex form consists of two subunits: Sec13‐dhfrp and a 150 kDa protein (p150). Native immunoprecipitation experiments confirm that Sec13p exists in a complex with p150 in wild type cells. Functional analysis supports a role for both subunits in protein transport. Vesicle budding from the ER in a cell‐free reaction is inhibited by Fab antibody fragments directed against either Sec13p or p150. The purified Sec13‐dhfrp/p150 complex, but not the Sec13‐dhfrp monomer, in combination with two other pure protein fractions (Sar1p and a Sec23/Sec24 protein complex) satisfies the requirement for cytosol in a cell‐free vesicle budding reaction. The vesicles formed with the purified protein fractions are competent to fuse with the Golgi and are biochemically distinct from the ER membrane fraction from which they derive.Keywords
This publication has 36 references indexed in Scilit:
- Requirement for a GTPase-Activating Protein in Vesicle Budding from the Endoplasmic ReticulumScience, 1993
- SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex.The Journal of cell biology, 1992
- Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.The Journal of cell biology, 1991
- Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles.The Journal of cell biology, 1991
- Dissection of a single round of vesicular transport: Sequential intermediates for intercisternal movement in the Golgi stackCell, 1989
- Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant.The Journal of cell biology, 1988
- Semi-intact cells permeable to macromolecules: Use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complexCell, 1987
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Intracellular Aspects of the Process of Protein SynthesisScience, 1975
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970