Cloning and partial characterization of the proteasome S4 ATPase from Plasmodium falciparum

The ATP-ubiquitin-proteasome pathway mediates the nonlysosomal degradation of cytosolic proteins in eukaryotic cells. The activities of this pathway h ave been shown to regulate cell growth and differentiation through modulati on of regulatory proteins. The proteasome is a large complex consisting of two multisubunit structures, the 20S and 19S(PA700) or P28 complexes, that combine to form the 26S particles. In this study, we describe the cloning o f a cDNA encoding the proteasome subunit 4 ATPase homologue from Plasmodium falciparum (PFS4). Analysis of the PFS4 cDNA sequence shows an open readin g frame encoding a deduced protein of 455 amino acids. Moreover, comparison of PFS4 cDNA sequence to that of genomic fragments encoding PFS4 showed id entical sequences with no detectable introns. Database searches revealed a high sequence identity to those of rice, yeast, mouse, Drosophila, and huma n S4 ATPases. However, PFS4 contains two unique inserts of nine and seven a mino acid residues in the N-terminal domain. Interestingly only the rice S4 contains the latter (seven amino acids) insert with four identical amino a cids. In vitro expression of the full-length cDNA encoding the PFS4, using a transcription-translation-coupled reticulocyte lysate, shows a 50-kDa [S- 35]methionine-labeled protein which was immunoprecipitated with PFS4 anti-p eptide antiserum. Southern blot analysis of genomic DNA digests shows a sin gle gene copy of PFS4 in P. falciparum. Of interest was the effect of the p roteasome-specific natural product, lactacystin, on the growth of the paras ite, with IC50 values of 0.6-0.92 mu M. The latter IC50 values of lactacyst in for different clones of P. falciparum are comparable to those obtained f or mammalian cell lines (0.65 mu M), suggesting the presence of a conserved proteasome complex. Moreover, lactacystin was equally toxic to drug-sensit ive and resistant parasites. (C) 1999 Academic Press.