Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes.

Abstract

Abstract. Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using afffinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver ceils carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

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

“…Representative absorbance spectra showed that the absorbance maximum of Trp in aqueous solution was 279 nm ( Figure 3A). In contrast, absorbance maxima of Trp in both proSCP-2 and SCP-2 were red-shifted to 283 nm ( Figure 3A), consistent with the single 50 Trp residue in proSCP-2 or SCP-2 each residing in a more nonpolar environment than the free Trp amino acid in aqueous solution (40). Finally, the maximal absorbance intensity of proSCP-2 was significantly higher than that of SCP-2 ( Figure 3A), suggesting the 50 Trp residue in proSCP-2 was located in a different microenvironment than was 50 Trp in SCP-2.…”

“…To this end, 50 Figure 4A). Further analysis of the acrylamide quenching data ( Figure 4B) yielded a dynamic quenching constant, K D , for 50 Trp in proSCP-2 that was 1.8-fold higher (P < 0.001) than that calculated for 50 Trp in SCP-2 ( Figure 4C). No static quenching component could be resolved for SCP-2; however, both proSCP-2 and free Trp in solution had calculated K S values that were statistically equivalent ( Figure 4C).…”

“…To further probe the 50 Trp microenvironment in proSCP-2 and SCP-2, fluorescence emission spectra (excitation at 280 nm) were obtained of each protein as well as an aqueous solution of the free Trp amino acid in aqueous buffer. Free Trp in solution exhibited the highest fluorescence emission intensity and had the longest emission maximum wavelength (356 nm) of the three species examined (Figue 3B), consistent with the highest exposure to the aqueous environment (41).…”

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

“…Mature SCP-2 can be generated by post-translational C-terminal proteolysis of either pro-SCP-2 or SCP-x [7,8]. Also known as non-specific lipid transfer protein because it recognizes phospholipids, fatty acids, and fatty acyl CoAs in addition to cholesterol, SCP-2 is found in several subcellular compartments, most prominently in peroxisomes, but also in mitochondria, lysosomes, and cytosol [7,9]. Multiple site location is consistent with the idea that SCP-2 is involved in lipid trafficking between compartments for key metabolic requirements, e.g.…”

The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity

“…However, ira. muno-electronmicroscopic labeling studies [17][18][19] as well as subcellular fractionation studies [20,21] have indicated that nsL-TP is not strictly peroxisomal. On the other hand, a specific relationship between nsl..-TP and peroxisomes is supported by the observations that nsL-TP is absent from Chinese hamster ovary (CHO) cells deficient in peroxisomes, and from Zellweger liver [20,22].…”