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The structure of Sec12 implicates potassium ion coordination in Sar1 activation.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2012 Dec 21; Vol. 287 (52), pp. 43599-606. Date of Electronic Publication: 2012 Oct 29. - Publication Year :
- 2012
-
Abstract
- Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the "K loop," which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.
- Subjects :
- COP-Coated Vesicles chemistry
COP-Coated Vesicles genetics
COP-Coated Vesicles metabolism
Cations, Monovalent chemistry
Cations, Monovalent metabolism
Crystallography, X-Ray
Guanine Nucleotide Exchange Factors genetics
Guanine Nucleotide Exchange Factors metabolism
Membrane Glycoproteins genetics
Membrane Glycoproteins metabolism
Monomeric GTP-Binding Proteins genetics
Monomeric GTP-Binding Proteins metabolism
Potassium metabolism
Protein Structure, Secondary
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae metabolism
Saccharomyces cerevisiae Proteins genetics
Saccharomyces cerevisiae Proteins metabolism
Structure-Activity Relationship
Vesicular Transport Proteins genetics
Vesicular Transport Proteins metabolism
Guanine Nucleotide Exchange Factors chemistry
Membrane Glycoproteins chemistry
Monomeric GTP-Binding Proteins chemistry
Potassium chemistry
Saccharomyces cerevisiae chemistry
Saccharomyces cerevisiae Proteins chemistry
Vesicular Transport Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 287
- Issue :
- 52
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 23109340
- Full Text :
- https://doi.org/10.1074/jbc.M112.420141