Biogenesis of nonspecific lipid transfer protein and sterol carrier protein x: studies using peroxisome assembly-defective pex cell mutants.
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.
Otera
H
H
Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan.
Nishimura
M
M
Setoguchi
K
K
Mori
T
T
Fujiki
Y
Y
D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D010940:Plant Proteins; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*
D002352:Carrier Proteins; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D013261:Sterols; C072087:sterol carrier proteins; D000101:Acetyl-CoA C-Acetyltransferase