Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops.

E Baumgart,T Hashimoto,A Völkl,H D Fahimi

doi:10.1083/jcb.108.6.2221

Abstract

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.

Highlights

It has been shown that the targeting signal for the import of proteins into POs may be located at the carboxy-terminal end of the PO proteins [28, 29] and in the amino-terminal part or even in the midportion [63], and it has been suggested that the proteins may be bound to a receptor before the ATP-dependent translocation over the PO membrane [38]
In this study we have used a new hypocholesterolemic drug BM 15766, which has a well-defined target in the pathway of cholesterol biosynthesis [3, 4]
The results indicate that those membranous structures which are associated with proliferating POs are heavily labeled with the antibody against 70-kD PO membrane proteins (PMP), indicating that they belong to the PO membrane system

Summary

Introduction

It has been shown that the targeting signal for the import of proteins into POs may be located at the carboxy-terminal end of the PO proteins [28, 29] and in the amino-terminal part or even in the midportion [63], and it has been suggested that the proteins may be bound to a receptor before the ATP-dependent translocation over the PO membrane [38]. The early observations ofthe close morphological association of POs with the smooth ER (SER) [51] led to the suggestion that POs may arise by budding from the ER [19, 52] This concept has been abandoned in the mean time, the exact nature and function of membranous structures seen occasionally attached to or associated with the PO membranes remains to be elucidated. Recent three-dimensional reconstruction studies ofultrathin sections have revealed that some of these structures are interconnecting bridges between two or several spherical POs [27, 74] These findings are consistent with the concept of a distinct intracellular compartment containing the PO matrix proteins [54] referred to as "the PO reticulum concept" [42]. Electrotransfer onto nitrocellulose sheets was accomplished at 30 V for 60 min

Methods

Results

Conclusion