title: Structure and function of the sterol carrier protein-2 N-terminal presequence.
abstract: Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, and fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2's affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts and caveolae (AF488-CTB); 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488 antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.
authors.affiliation: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, Texas 77843-4466, USA.
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Martin
authors.forename: Heather A
authors.initials: HA
authors.lastname: Hostetler
authors.forename: Avery L
authors.initials: AL
authors.lastname: McIntosh
authors.forename: Shane E
authors.initials: SE
authors.lastname: Tichy
authors.forename: Brad J
authors.initials: BJ
authors.lastname: Williams
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Berg
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Spencer
authors.forename: Judith
authors.initials: J
authors.lastname: Ball
authors.forename: Ann B
authors.initials: AB
authors.lastname: Kier
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Schroeder
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008024:Ligands; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D011498:Protein Precursors; D011994:Recombinant Proteins; C072087:sterol carrier proteins; D014364:Tryptophan