Biogenesis of Nonspecific Lipid Transfer Protein and Sterol Carrier Protein x

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 prensLTP.

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…By retention, stabilisation and presentation of fatty acid oxidation intermediates suitable for catabolism in the peroxisome, coupled to rejection of carnitine derivatives, SCP2 could significantly increase the efficiency of cellular fatty acid oxidation. One in vivo study has indicated that there is a pool of preSCP2 in the cytosol [9]. In this study we have not been able to distinguish any significant difference in activity of preSCP2 or mSCP2 towards fatty acyl carnitines in vitro, thus there is no basis for suspecting that the presence of a cytosolic concentration of preSCP2 will interfere with the uptake of fatty acyl carnitine derivatives by the mitochondrion.…”

“…The NR-BA assay was previously used to demonstrate the 2:1 binding of linoleoyl [16,34] between the isoforms. The presequence and PTS1 are thus implicated in regulating SCP2 localization [9,34]. The physiological relevance of the lack of LCFA-carnitine binding capacity of SCP2 may relate to the dual role of the peroxisome and the mitochondrion in fatty acid b-oxidation.…”

“…Further to this, different isoforms of the same gene product can be found in a single cell-the SCPx gene can produce an alternate transcript encoding 15 kDa preSCP2 [1,7], identical to the C-terminal domain of SCPx. Both SCPx and preSCP2 carry a type 1 peroxisomal targeting signal (PTS1) tripeptide at the extreme C-terminus [8] and both appear to be substrates for intraperoxisomal proteolytic cleavage to yield 13 kDa mature mSCP2 [1,9,10].…”

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

“…Although the rapid segmental mobility of the 20-amino acid N-terminal presequence on the NMR and X-ray time scale has led to the interpretation that these regions are unstructured, this does not necessarily exclude the presence of secondary structure elements within these mobile segments. On the contrary, Chou-Fasman modeling of the 20-amino acid N-terminal presequence suggests the presence of both α-helix and β-sheet structures therein (45). Likewise secondary structure prediction based on the program PHDsec suggests the presence of an α-helix in the presequence (46).…”

“…However, on the basis of secondary structure modeling of the 20-amino acid presequence (Figure 1), it has been suggested that the presequence contains a small α-helical element from amino acid −17 to −15 and a β-sheet from amino acid −8 to −3 (45). To begin to address this possibility and obtain molecular details of the structure of these polypeptide sequences, a series of proSCP-2 and SCP-2 N-terminal peptides was synthesized for secondary structure analysis by circular dichroism:…”