ABSTRACT
Yeast Sec14p acts as a phosphatidylinositol/phosphatidylcholine-transfer protein in vitro. In vivo, it is essential in promoting Golgi secretory function. Products of five genes named SFH1-SFH5 (Sec Fourteen Homologues 1-5) exhibit significant sequence homology to Sec14p and together they form the Sec14p family of lipid-transfer proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism and membrane trafficking.
Subject(s)
Phosphatidylinositols/metabolism , Phospholipid Transfer Proteins/physiology , Saccharomyces cerevisiae/physiology , Sequence Homology, Amino Acid , Multigene Family , Phosphatidylinositols/chemistry , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/geneticsABSTRACT
Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha-helix, 7-fold more beta-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar K(d) values) in the order: cholesterol straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of l-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, l-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.
Subject(s)
ATP-Binding Cassette Transporters , Carrier Proteins/chemistry , Peroxisomes/metabolism , Plant Proteins , Protein Precursors/chemistry , Animals , Blotting, Western , Carboxypeptidases/metabolism , Carboxypeptidases A , Carrier Proteins/physiology , Cell Line , Cell Membrane/metabolism , Cholesterol/metabolism , Circular Dichroism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Fatty Acids/metabolism , Fluorescent Antibody Technique , Fluorescent Antibody Technique, Indirect , Guanidine/pharmacology , Humans , Immunoblotting , Kinetics , Ligands , Membrane Proteins/metabolism , Microscopy, Confocal , Protein Folding , Protein Precursors/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Transfection , Tumor Cells, CulturedABSTRACT
A lipid transfer protein with a broad substrate specificity is associated with the peroxisomal membrane of the yeast Saccharomyces cerevisiae. The protein catalyzes in vitro the transfer of various phospholipids, phosphatidylinositol and phosphatidylserine being translocated at the highest rates. The transfer protein can be released from peroxisomal membranes by treatment with 0.25 M KCl and highly enriched using conventional chromatographic techniques. It is inactivated by heat, detergents, divalent cations and proteinases. During various steps of purification this lipid transfer protein co-fractionated with peroxisomal acyl-CoA oxidase (Pox1p). In a pox1 disruptant peroxisomal lipid transfer activity was still present, although at a reduced level. The peroxisomal lipid transfer protein from the pox1 mutant exhibited different chromatographic properties as compared to the wild-type strain suggesting that acyl-CoA oxidase and the peroxisomal lipid transfer protein may from a complex.
Subject(s)
Carrier Proteins/metabolism , Fungal Proteins/metabolism , Microbodies/chemistry , Plant Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Acyl-CoA Oxidase , Biological Transport , Carrier Proteins/chemistry , Fungal Proteins/chemistry , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Microbodies/metabolism , Oxidoreductases/metabolism , Protein BindingABSTRACT
An antibody raised against human sterol carrier protein 2 (SCP-2) crossreacts with two yeast peroxisomal proteins. These proteins have apparent molecular weights of 35 and 58 kDa. Subfractionation of peroxisomes revealed that the 58 kDa species is a soluble matrix protein, whereas the 35 kDa protein is membrane bound. Treatment of isolated peroxisomal membranes with 0.25 M KCl released the 35 kDa crossreactive protein into the soluble supernatant. However, lipid transfer activity could be attributed neither to the 35 kDa nor to the 58 kDa protein.
