The Synthesis and Turnover of Rat Liver Peroxisomes

Abstract. Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring PronLeu polymorphism and a PHi-specific GlylToArg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly~70Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the PronLeu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro11Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import. BOTH mitochondrial and peroxisomal precursor proteins are synthesized on free polyribosomes in the cytosol and are imported posttranslationally into their respective organelles (17,25). Despite these similarities, many studies suggest that the targeting information and structural requirements for mitochondrial and peroxisomal protein import are very different. Most mitochondrial precursor proteins are synthesized with NH2-termi-

Section: Discussionmentioning

confidence: 99%

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1.

Leiper

Oatey

Danpure

1996

“…In addition to the above mentioned membrane proteins, ferritin (25), and catalase (26,27), some microsomai components, such as NADPH-cytochrome c reductase (28) and cytochrome P-450 (29), are believed to be synthesized at least partially on free ribosomes. The ribosomes themselves rapidly exchange their proteins with equivalent proteins in the cytoplasm (30).…”

Section: Discussionmentioning

confidence: 99%

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

Elhammer¹,

Svensson²,

Autuori³

et al. 1975

The presence in the Golgi fraction ofglycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [l'C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer being most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.

“…pages 507-524 scribed in the preceding paper (22) . The animals were sacrificed at various times thereafter and their livers rapidly removed and chilled .…”

Section: Experimental Designmentioning

confidence: 99%

“…The PNS was fractionated as described below, and the fractions, as well as the original PNS, were assayed for protein, RNA, marker enzymes, TCA-precipitable radioactivity, and immunoprecipitable radioactivity. Immunoprecipitation was carried out exactly as described in the preceding paper (22), both on the whole fractions to yield whole fraction immunoprecipitates (WFI), and after partial purification of catalase to yield purified catalase immunoprecipitates (PCI) . Only goat globulins were used .…”

Section: Experimental Designmentioning

confidence: 99%

“…It has been shown in the preceding paper of this series (22) that rat liver catalase is synthesized by way of two distinct intermediates, one without heme and having a turnover time of about 49 min, the other containing heme and having a turnover time of about 17 min . The topology of this process has been investigated in the present experiments.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The Synthesis and Turnover of Rat Liver Peroxisomes

Lazarow

Duve

1973

Self Cite

194

The subcellular distribution of the biosynthetic intermediates of catalase was studied in the livers of rats receiving a mixture of [3H]leucine and [14C]δ-aminolevulinic acid by intraportal injection. Postnuclear supernates were fractionated by a one-step gradient centrifugation technique that separates the main subcellular organelles, partly on the basis of size, and partly on the basis of density. Labeled catalase and its biosynthetic intermediates were separated from the gradient fractions by immunoprecipitation, and the distributions of radioactivity were compared with those of marker enzymes. The results show that catalase protein is synthesized outside the peroxisomes, but rapidly appears in these particles, mostly still in the form of the first hemeless biosynthetic intermediate. Addition of heme and completion of the catalase molecule take place within the peroxisomes. During the first 15 min after [3H]leucine administration, more than half of the newly formed first intermediate was recovered in the supernatant fraction, where it was found to exist as an aposubunit of about 60,000 molecular weight.