Abstract
Nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We investigated which of these two potential topogenic sequences functions in biogenesis of nsLTP and SCPx. Morphological and biochemical analyses, making use of Chinese hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective pex7, as well as green fluorescent protein chimeras, revealed that both pre-nsLTP and SCPx are imported into peroxisomes by the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains in the cytosol, as assessed by subcellular fractionation of the wild-type Chinese hamster ovary cells. In an in vitro binding assay, only mature nsLTP, but not pre-nsLTP, from the cell lysates interacted with the Pex5p. It is likely, therefore, that modulation of the C-terminal PTS1 by the presequence gives rise to cytoplasmic localization of pre-nsLTP.
Highlights
Nonspecific lipid transfer protein with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus
Two types of cDNA for Nonspecific lipid transfer protein (nsLTP) of rat liver were cloned (6 – 8), one encoding 15-kDa pre-nsLTP consisting of 143 amino acids, a precursor form of 123-amino acid mature nsLTP [9, 10], and the other coding for a 547-amino acid protein with a mass of 59 kDa, termed sterol carrier protein x (SCPx)
To address the underlying mechanisms by which nsLTP and Sterol carrier protein x (SCPx) are transported into peroxisomes, we investigated biogenesis of these proteins at morphological, as well as biochemical, levels using several types of peroxisome biogenesis-defective cell mutants and the PTS1 receptor Pex5p
Summary
Nonspecific lipid transfer protein (nsLTP; called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We report that import of nsLTP and SCPx was affected in pex mutant of Chinese hamster ovary (CHO) cells deficient in import of PTS1 and PTS2, whereas in PTS2 import-defective pex mutant both proteins were imported into peroxisomes as effi-.
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