Novel Peroxisomal Populations in Subcellular Fractions from Rat Liver

doi:10.1074/jbc.270.12.6949

Novel Peroxisomal Populations in Subcellular Fractions from Rat Liver

IMPLICATIONS FOR PEROXISOME STRUCTURE AND BIOGENESIS (*)

From the (1)Department of Metabolic Research, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, Stockholm and the
(2)Clinical Research Center, Huddinge Hospital, Huddinge and
(3)Department of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, Huddinge, Sweden

^§ To whom correspondence should be addressed:
Dept. of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
Tel.: 46-8-746-1601; Fax: 46-8-746-1698.

Abstract

According to current concepts, new peroxisomes are formed by division of pre-existing peroxisomes or by budding from a peroxisomal reticulum. Recent cytochemical and biochemical data indicate that protein content in peroxisomes are heterogenous and that import of newly synthesized proteins may be restricted to certain protein import-competent peroxisomal subcompartments (Yamamoto, K., and Fahimi, H. D.(1987) J. Cell Biol. 105, 713-722; Heinemann, P., and Just, W. W.(1992) FEBS Lett. 300, 179-182; Lüers, G., Hashimoto, T., Fahimi, H. D., and Völkl, A.(1993) J. Cell Biol. 121, 1271-1280).

We have observed that substantial amounts of peroxisomal proteins are found together with “microsomes” (100,000 × g pellet) after subcellular fractionation of rat liver homogenates. In this study we have investigated the origin of these peroxisomal proteins by modified gradient centrifugation procedures in Nycodenz and by analysis of enzyme activity distributions, Western blotting, and immunoelectron microscopy. It is concluded that much of this material is confined to novel populations of “peroxisomes.” Immunocytochemistry on gradient fractions showed that some vesicles were enriched in acyl-CoA oxidase and peroxisomal multifunctional enzyme (“catalase-negative”) whereas others were enriched in catalase and thiolase (“acyl-CoA oxidase-negative”). Double immunolabeling experiments verified the strong heterogeneity in the protein contents of these vesicles and also identified peroxisomes varying in size from about 0.5 μm (“normal peroxisomes”) to extremely small vesicles of less than 100 nm in diameter. The possibility that these vesicles may be related to different subcompartments of a larger peroxisomal structure involved in protein import and biogenesis will be discussed.

Footnotes

↵* This work was supported by grants from the Swedish Natural Science Research Council, The Bank of Sweden Tercentenary Foundation, “Magnus Bergvalls Stiftelse,” “Lars Hiertas Minne,” “Tore Nilsons Fond för Medicinsk Forskning,” “Hierta-Retzius' fond för vetenskaplig forskning,” and the “Ax:son Johnsons Stiftelse.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

Aox

acyl-CoA oxidase

DEHP

di(2-ethylhexyl)phthalate

MFE

peroxisomal multifunctional enzyme

PBB

phosphate-buffered bovine serum albumin

PMP70

the 70-kDa peroxisomal integral membrane protein

HM

heavy mitochondrial fraction

LM

light mitochondrial fraction

SKL

Ser-Lys-Leu

PTS

peroxisomal targeting signal

DAB

3,3′-diaminobenzidine. Note: from the distributions of marker enzymes we conclude that nearly all of the 3-hydroxyacyl-CoA dehydrogenase activity found in the microsomal fraction is due to the peroxisomal multifunctional enzyme (MFE), expressing hydratase/dehydrogenase/isomerase activity(69). The abbreviation MFE is used to denote this protein when detected with a monospecific antibody in Western blotting and immunoelectron microscopy, whereas 3-hydroxyacyl-CoA dehydrogenase is used when the activity is measured in the gradient fractions.
↵²T. Svensson, M. Wilcke, S. Alexson, H. Häyrinen, R. Sormunen, and K. Hiltunen, submitted for publication.
↵³A. Messing-Eriksson and S. Alexson, unpublished results.
- Received April 4, 1994.
- Revision received September 13, 1994.