title: Crystallization and preliminary X-ray diffraction analysis of Sfh3, a member of the Sec14 protein superfamily.
abstract: Sec14 is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the yeast Saccharomyces cerevisiae and is the founding member of the Sec14 protein superfamily. Recent functional data suggest that Sec14 functions as a nanoreactor for PtdCho-regulated presentation of PtdIns to PtdIns kinase to affect membrane trafficking. Extrapolation of this concept to other members of the Sec14 superfamily suggests a mechanism by which a comprehensive cohort of Sec14-like nanoreactors sense correspondingly diverse pools of lipid metabolites. In turn, metabolic information is translated to signaling circuits driven by phosphoinositide metabolism. Sfh3, one of five Sec14 homologs in yeast, exhibits several interesting functional features, including its unique localization to lipid particles and microsomes. This localization forecasts novel regulatory interfaces between neutral lipid metabolism and phosphoinositide signaling. To launch a detailed structural and functional characterization of Sfh3, the recombinant protein was purified to homogeneity, diffraction-quality crystals were produced and a native X-ray data set was collected to 2.2 Å resolution. To aid in phasing, SAD X-ray diffraction data were collected to 1.93 Å resolution from an SeMet-labeled crystal at the Southeast Regional Collaborative Access Team at the Advanced Photon Source. Here, the cloning and purification of Sfh3 and the preliminary diffraction of Sfh3 crystals are reported, enabling structural analyses that are expected to reveal novel principles governing ligand binding and functional specificity for Sec14-superfamily proteins.
authors.affiliation: Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090, USA. jihui_ren@med.unc.edu
authors.forename: Jihui
authors.initials: J
authors.lastname: Ren
authors.forename: Gabriel
authors.initials: G
authors.lastname: Schaaf
authors.forename: Vytas A
authors.initials: VA
authors.lastname: Bankaitis
authors.forename: Eric A
authors.initials: EA
authors.lastname: Ortlund
authors.forename: Manish C
authors.initials: MC
authors.lastname: Pathak
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D049294:Phospholipid Transfer Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D016415:Sequence Alignment
chemical_list: C483431:PDR16 protein, S cerevisiae; D049294:Phospholipid Transfer Proteins; D029701:Saccharomyces cerevisiae Proteins
references.citation: J Cell Biol. 2002 Apr 1;157(1):63-77
references.pmid: 11916983
references.citation: Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14713-8
references.pmid: 14657365
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Nature. 1998 Jan 29;391(6666):506-10
references.pmid: 9461221
references.citation: Trends Biochem Sci. 2010 Mar;35(3):150-60
references.pmid: 19926291
references.citation: Traffic. 2005 Dec;6(12):1157-72
references.pmid: 16262726
references.citation: Mol Biol Cell. 2002 Jul;13(7):2193-206
references.pmid: 12134061
references.citation: J Cell Biol. 1994 Feb;124(3):273-87
references.pmid: 8294512
references.citation: Cell. 1991 Feb 22;64(4):789-800
references.pmid: 1997207
references.citation: EMBO J. 1990 May;9(5):1665-72
references.pmid: 2184035
references.citation: Nature. 1990 Oct 11;347(6293):561-2
references.pmid: 2215682
references.citation: J Mol Biol. 2003 Sep 26;332(4):759-65
references.pmid: 12972248
references.citation: EMBO Rep. 2006 Feb;7(2):174-9
references.pmid: 16397625
references.citation: Mol Biol Cell. 2000 Jun;11(6):1989-2005
references.pmid: 10848624
references.citation: Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12346-51
references.pmid: 9770489
references.citation: Mol Cell. 2008 Feb 1;29(2):191-206
references.pmid: 18243114
references.citation: J Mol Biol. 2007 Feb 16;366(2):551-62
references.pmid: 17187824
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3497-500
references.pmid: 12824352
references.citation: Mol Cell. 1999 Aug;4(2):187-97
references.pmid: 10488334
references.citation: J Struct Biol. 2003 Apr;142(1):170-9
references.pmid: 12718929
references.citation: J Mol Biol. 2003 Aug 15;331(3):725-34
references.pmid: 12899840
references.citation: Biochim Biophys Acta. 2007 Jun;1771(6):737-45
references.pmid: 17395532