| Structural highlights
Function
[SEC12_YEAST] Guanine nucleotide-exchange factor (GEF) required for the formation or budding of transport vesicles from the ER. This function involves the cytoplasmic domain of the protein, which is thought to interact with the small GTP-binding protein SAR1. Required for autophagy.[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Publication Abstract from PubMed
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 ER membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.35 A resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a beta 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, reveal 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.
The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.,McMahon C, Studer SM, Clendinen C, Dann GP, Jeffrey PD, Hughson FM J Biol Chem. 2012 Oct 29. PMID:23109340[17]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Ishihara N, Hamasaki M, Yokota S, Suzuki K, Kamada Y, Kihara A, Yoshimori T, Noda T, Ohsumi Y. Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion. Mol Biol Cell. 2001 Nov;12(11):3690-702. PMID:11694599
- ↑ Hamasaki M, Noda T, Ohsumi Y. The early secretory pathway contributes to autophagy in yeast. Cell Struct Funct. 2003 Feb;28(1):49-54. PMID:12655150
- ↑ Hardwick KG, Boothroyd JC, Rudner AD, Pelham HR. Genes that allow yeast cells to grow in the absence of the HDEL receptor. EMBO J. 1992 Nov;11(11):4187-95. PMID:1327759
- ↑ Futai E, Schekman R. Purification and functional properties of yeast Sec12 GEF. Methods Enzymol. 2005;404:74-82. PMID:16413259 doi:https://dx.doi.org/10.1016/S0076-6879(05)04008-5
- ↑ Rexach MF, Schekman RW. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219-29. PMID:1649197
- ↑ d'Enfert C, Wuestehube LJ, Lila T, Schekman R. Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER. J Cell Biol. 1991 Aug;114(4):663-70. PMID:1907973
- ↑ Oka T, Nishikawa S, Nakano A. Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport. J Cell Biol. 1991 Aug;114(4):671-9. PMID:1907974
- ↑ d'Enfert C, Barlowe C, Nishikawa S, Nakano A, Schekman R. Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum. Mol Cell Biol. 1991 Nov;11(11):5727-34. PMID:1922074
- ↑ Nakano A, Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677-91. PMID:2512296
- ↑ Nakano A, Brada D, Schekman R. A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J Cell Biol. 1988 Sep;107(3):851-63. PMID:3047151
- ↑ Novick P, Field C, Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205-15. PMID:6996832
- ↑ Nishikawa S, Hirata A, Nakano A. Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies. Mol Biol Cell. 1994 Oct;5(10):1129-43. PMID:7865879
- ↑ Oka T, Nakano A. Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast. J Cell Biol. 1994 Feb;124(4):425-34. PMID:8106544
- ↑ Barlowe C, Schekman R. SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature. 1993 Sep 23;365(6444):347-9. PMID:8377826 doi:https://dx.doi.org/10.1038/365347a0
- ↑ Barlowe C, Schekman R. Expression, purification, and assay of Sec12p: a Sar1p-specific GDP dissociation stimulator. Methods Enzymol. 1995;257:98-106. PMID:8583945
- ↑ Campbell JL, Schekman R. Selective packaging of cargo molecules into endoplasmic reticulum-derived COPII vesicles. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):837-42. PMID:9023343
- ↑ McMahon C, Studer SM, Clendinen C, Dann GP, Jeffrey PD, Hughson FM. The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation. J Biol Chem. 2012 Oct 29. PMID:23109340 doi:https://dx.doi.org/10.1074/jbc.M112.420141
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