This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Small, G M Articles by Lazarow, P B Search for Related Content PubMed PubMed Citation Articles by Small, G M Articles by Lazarow, P B

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

ABSTRACT

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

This article has been cited by other articles:

• Lin-Cereghino, G. P., Godfrey, L., de la Cruz, B. J., Johnson, S., Khuongsathiene, S., Tolstorukov, I., Yan, M., Lin-Cereghino, J., Veenhuis, M., Subramani, S., Cregg, J. M. (2006). Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris. Mol. Cell. Biol. 26: 883-897 [Abstract] [Full Text]