Pro-sterol carrier protein-2. Role of the N-terminal presequence in structure, function, and peroxisomal targeting

F. Schroeder, A. Frolov, O. Starodub, B. B. Atshaves, William Russell, A. Petrescu, H. Huang, A. M. Gallegos, A. McIntosh, D. Tahotna, D. H. Russell, J. T. Billheimer, C. L. Baum, A. B. Kier

Research output: Contribution to journalArticle

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Abstract

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 α-helix, 7-fold more β-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.

Original languageEnglish (US)
Pages (from-to)25547-25555
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number33
DOIs
StatePublished - Aug 18 2000
Externally publishedYes

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Fatty Acids
Complementary DNA
Carboxypeptidases A
Amino Acids
Confocal microscopy
Guanidine
Sterols
Cell membranes
Peroxisomes
Protein Precursors
Protein Stability
Binding Sites
Cholesterol
Spectroscopy
Circular Dichroism
Ligands
Scanning
Lipid Metabolism
Confocal Microscopy
Fluorescent Antibody Technique

ASJC Scopus subject areas

  • Biochemistry

Cite this

Pro-sterol carrier protein-2. Role of the N-terminal presequence in structure, function, and peroxisomal targeting. / Schroeder, F.; Frolov, A.; Starodub, O.; Atshaves, B. B.; Russell, William; Petrescu, A.; Huang, H.; Gallegos, A. M.; McIntosh, A.; Tahotna, D.; Russell, D. H.; Billheimer, J. T.; Baum, C. L.; Kier, A. B.

In: Journal of Biological Chemistry, Vol. 275, No. 33, 18.08.2000, p. 25547-25555.

Research output: Contribution to journalArticle

Schroeder, F, Frolov, A, Starodub, O, Atshaves, BB, Russell, W, Petrescu, A, Huang, H, Gallegos, AM, McIntosh, A, Tahotna, D, Russell, DH, Billheimer, JT, Baum, CL & Kier, AB 2000, 'Pro-sterol carrier protein-2. Role of the N-terminal presequence in structure, function, and peroxisomal targeting', Journal of Biological Chemistry, vol. 275, no. 33, pp. 25547-25555. https://doi.org/10.1074/jbc.M000431200
Schroeder, F. ; Frolov, A. ; Starodub, O. ; Atshaves, B. B. ; Russell, William ; Petrescu, A. ; Huang, H. ; Gallegos, A. M. ; McIntosh, A. ; Tahotna, D. ; Russell, D. H. ; Billheimer, J. T. ; Baum, C. L. ; Kier, A. B. / Pro-sterol carrier protein-2. Role of the N-terminal presequence in structure, function, and peroxisomal targeting. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 33. pp. 25547-25555.
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abstract = "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 α-helix, 7-fold more β-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.",
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T1 - Pro-sterol carrier protein-2. Role of the N-terminal presequence in structure, function, and peroxisomal targeting

AU - Schroeder, F.

AU - Frolov, A.

AU - Starodub, O.

AU - Atshaves, B. B.

AU - Russell, William

AU - Petrescu, A.

AU - Huang, H.

AU - Gallegos, A. M.

AU - McIntosh, A.

AU - Tahotna, D.

AU - Russell, D. H.

AU - Billheimer, J. T.

AU - Baum, C. L.

AU - Kier, A. B.

PY - 2000/8/18

Y1 - 2000/8/18

N2 - 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 α-helix, 7-fold more β-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.

AB - 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 α-helix, 7-fold more β-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.

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