Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that the se vesicles were enriched in acid phosphatase and cysteine protease activit ies. An important virulence factor, a 27-kDa cysteine protease, was also en riched in early and late endosomes of E. histolytica. These data suggest th at E. histolytica hydrolases reside in compartments that are part of or com municate with the endosomal pathway. To begin to identify the role of Rab G TPases in E. histolytica, an oligonucleotide approach was employed to scree n an E. histolytica cDNA library for genes encoding Rab-like proteins, cDNA s encoding a Rab11-like protein (EhRab11) and a novel Rab protein (EhRabA) were isolated and characterized. The EhRab11 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 23.2 kDa. Phylo genetic analysis and alignment of EhRab11 with other Rab proteins demonstra ted that EhRabll shared significant homology at the amino acid level with R ab11-like proteins from a number of other eukaryotes, suggesting that EhRab 11 is a Rab11 homolog for E. histolytica. The EhRabA clone predicts a polyp eptide of 219 amino acids with a molecular mass of at least 24.5 kDa. EhRab A shared only limited homology at the amino acid level with other Rab prote ins, suggesting that it is a novel member of this family of CTP-binding pro teins. Finally, Western blot analysis demonstrated that EhRabll and a previ ously described Rab7-like GTPase from E. histolytica was enriched in magnet ically purified endosomal compartments of this organism. (C) 1999 Elsevier Science B.V. All rights reserved.