No abstract

Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy‐termini. One such protein is the peroxisomal 3‐ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600–4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino‐terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino‐terminal or internal locations and is distinct from the C‐terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.

Two types of ~ptid¢ signals are known to independently target proteins into the peroxisomal matrix. One of these is a consensus C-terminal tripe, pride which is conserved in many microbody proteins derived from diverse Slmcies. The second signal is an N-terminal sequence found in a small subset of peroxisomal proteins. We have tested 18 possible variants of the consensus tripeptic[e targeting signal for their ability to facilitate the transport of a cytosolie passenger protein, ehloramphenieol aeetyltransferase, into peroxisomes of monkey kidney cells. Our results reveal the presence of a hierarchy of preferred amino acid substitutions at each position of the tripeptide.

Communicated by P.BorstTo determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share -45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.

Abstract. Trypanosomes compartmentalize most of