Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5Ј-[␥-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface. INTRODUCTIONProteins internalized via receptor-mediated endocytosis are rapidly transported via clathrin-coated vesicles to endosomal structures located in the periphery of the cell known as early or sorting endosomes (Gruenberg and Maxfield, 1995;Mellman, 1996;Clague, 1998). From there, ligands and their cognate receptor as well as fluid material are delivered via late endosomes to lysosomes where they are degraded. Some of the receptors, including the transferrin (Tf) receptor (TfR), are recycled back to the cell surface from the peripheral endosomes, either directly or indirectly, via pericentriolar recycling endosomes.One of the unsolved issues is the mechanism by which those plasma membrane proteins that are not degraded are transported out of the sorting endosomes. It is not clear whether the transport vehicles are tubules or vesicles. It is also not known in molecular terms how recycling membrane proteins are sorted from each other in the sorting endosome. For example, there is evidence that membrane proteins are transported to the recycling endosomes by default, following the bulk flow of lipids (Mayor et al., 1993). On the other hand, there has been circumstantial evidence to support a role for coated vesicles in the recycling of these proteins from endosomes. A number of coat proteins copurify with fractions enriched in early endosomes (Whitney et al., 1995).Electron microscopic studies also demonstrate budding profiles on endosomes, many containing clathrin and other coat proteins (Stoorvogel et al., 1996;Futter et al., 1998). Endo- somes from specialized neuroendocrine PC12 cells have also been shown to give rise to a subset of synap...
The ornithine aminotransferase (OAT) activity of mouse was found to be highest in the small intestine. The mitochondrial OAT from mouse small intestine was purified to homogeneity by the procedures including heat treatment, ammonium sulfate fractionation, octyl-S e p h a r o s e chromatography, and Sephadex G-150 gel filtration. Comparing to the amino acid sequence of mouse hepatic OAT, six N-terminal amino acid r e s i d u e s have been deleted in intestinal OAT. However, t h e subsequent sequence was identical with that of hepatic OAT. The molecular weights of both intestinal and hepatic OAT were estimated as 46 kDa by SDSgel electrophoresis and as 92 kDa by gel filtration, indicating that both native OATs are dimeric. Biochemical properties of intestinal OAT, such as molecular weight, pH optimum and K m values for Lornithine and -ketoglutarate, were similar to those of hepatic OAT. However, intestinal OAT was more labile than hepatic OAT to tryptic digestion. Keywords: ornithine aminotransferase; mouse; small intestine IntroductionOrnithine aminotransferase (OAT; L-ornithine:2-oxo-acid aminotransferase; EC 2.6.1.13) is a pyridoxal phosphaterequiring enzyme which catalyzes the reversible transamination of L-ornithine and a-ketoglutarate to glutamate and glutamic-γ-semialdehyde (its cyclized form being Δ 1 -pyrroline-5-carboxylate), and the latter product can be reversibly converted to no undeeline (Peraino and Pitot, 1963). OAT is expressed in nearly all mammalian tissues, including liver, kidney, brain, skeletal muscle, and eyes. OAT plays a role in arginine catabolism, proline biosynthesis, or de novo ornithine biosynthesis, depending on the tissue and the physiological circumstances (Mestichelli et al., 1979; Merril and Pitot,1983). Liver OAT is suggested to be involved in the ornithine synthesis for urea cycle while the kidney OAT participates in ornithine degradation (Herzfeld and Knox, 1968;Volpe et al. , 1969). Matsuzawa et al. (1994) suggested that the intestinal OAT may be involved in the ornithine supply to the liver, with the reversal of OAT reaction. In human, a genetic deficiency of OAT causes gyrate atrophy, an autosomal recessive degenerative disease of the choroid and retina of the eye that leads to blindness ( Valle et al., 1977;Kobayashi et al., 1995).OAT is known as a mitochondrial matrix enzyme and has been purified from various tissue sources, including rat liver, kidney, and brain (Matsuzawa et al., 1968;Sanada et al., 1970;Deshmukh, 1984) as well as human liver (Ohura et al., 1982). The mitochondrial OAT has been shown to be synthesized as a large precursor molecule with N-terminal leader peptide on cytoplasmic ribosome, which is then processed and becomes associated with the mitochondrion (Mueckler and Pitot, 1985). The amino acid sequence of OAT precursor protein was predicted from the nucleotide sequence of cDNA (Mueckler and Pitot, 1985;Inaga et al., 1986;Ramesh et al., 1986;Giometti et al., 1992). Recently it was reported that human kidney OAT had the same nucleotide sequenc...
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