Pro-sterol carrier protein-2: role of the N-terminal presequence in structure, function, and peroxisomal targeting.
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.
Schroeder
F
F
Department of Physiology and Pharmacology, Texas A & M University, College Station 77843-4466, USA. fschroeder@cvm.tamu.edu
Frolov
A
A
Starodub
O
O
Atshaves
B B
BB
Russell
W
W
Petrescu
A
A
Huang
H
H
Gallegos
A M
AM
McIntosh
A
A
Tahotna
D
D
Russell
D H
DH
Billheimer
J T
JT
Baum
C L
CL
Kier
A B
AB
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D019084:Fluorescent Antibody Technique, Indirect; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D020675:Peroxisomes / Q000378:metabolism*; D010940:Plant Proteins; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
C063305:ABCD3 protein, human; D018528:ATP-Binding Cassette Transporters; C489272:Abcd3 protein, rat; D002352:Carrier Proteins; D018076:DNA, Complementary; D005227:Fatty Acids; D008024:Ligands; D008565:Membrane Proteins; D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C072087:sterol carrier proteins; D002784:Cholesterol; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D019791:Guanidine