Abstract. Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the transGolgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures Ͻ 20 Њ C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.
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 x 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 microns ("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.
A common function of peroxisomes in eukaryotic cells is P-oxidation of fatty acids. In animal cells, P-oxidation is compartmentalized to peroxisomes and mitochondria. Although regulation of P-oxidation in mitochondria has been extensively studied, knowledge on its regulation in peroxisomes is still limited. We have considered the possibility that peroxisomes may contain acyl-CoA thioesterases with different substrate specificities that possibly regulate metabolism of different lipids by regulation of substrate availability. In the present study, we have investigated the presence of short-chain and long-chain acyl-CoA thioesterase activities in rat liver peroxisomes.Light-mitochondria1 fractions, enriched in peroxisomes, were fractionated by Nycodenz density gradient centrifugation and gradient fractions were analyzed for acyl-CoA thioesterase and marker enzyme distributions. Fractionation of livers from normal rats showed that most of the long-chain acyl-CoA thioesterase activity was localized in microsomes and mitochondria, and only low activity was found in fractions containing peroxisomes. The gradient distribution of propionyl-CoA thioesterase activity showed this activity to be localized mainly in mitochondria and in fractions possibly representing lysosomes, with a small peak of activity in peroxisomal fractions.Di(2-ethylhexy1)phthalate treatment induced the specific propionyl-CoA thioesterase activity approximately threefold in the peak mitochondrial fractions and about onefold in peroxisomal fractions ; the activity appeared to be almost exclusively localized to these organelles. The specific activity of myristoyl-CoA thioesterase was induced 1 -2-fold in peroxisomal peak fractions and more than 10-fold in the mitochondrial peak fraction, whereas it was unchanged in microsomes.The chain-length specificity of acyl-CoA thioesterase activity in isolated peroxisomes suggests that peroxisomes contain an inducible short-chain thioesterase active on C,-C, acyl-CoA species (possibly a 'propionyl-CoA' thioesterase). In addition, peroxisomes contain medium-chain to longchain thioesterase activity, probably due to separate enzymes based on the different chain-length specificities observed in peroxisomes from normal and di(2-ethylhexy1)phthalate-treated rats.A long-chain acyl-CoA thioesterase was partially purified from isolated peroxisomes and found to be active only on fatty-acyl-CoA species longer than octanoyl-CoA. The protein is apparently a monomer of about 40 kDa and clearly different from microsomal long-chain acyl-CoA thioesterase.An induction of this long-chain thioesterase may explain the observed change in chain-length specificity in peroxisomes isolated from normal and di(2-ethylhexy1)phthalate-treated rats.Possible physiological functions of these thioesterases are discussed.P-oxidation of fatty acids is compartmentalized to mitochondria and peroxisomes in animal cells. Peroxisomes are involved in the metabolism of various lipids (for review, see Lazarow and Moser, 1989) such as fatty acids (Lazarow and
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