A dominant negative mutation in the GIM1 gene of Leishmania donovani is responsible for defects in glycosomal protein localization

Kinetoplastid protozoa contain a unique microbody organelle called the glyc osome. Several important metabolic pathways are compartmentalized within th e glycosome that are found in the cytoplasm of higher eukaryotes. We have p reviously reported the identification of a Leishmania donovani cell line ca lled gim1-1, in which several normally glycosomal proteins are partially mi slocalized to the cytoplasm. The GIM1 gene complements the defect and resto res import of proteins to the glycosome. Here we demonstrate that GIM1 enco des an integral membrane protein of the glycosome. We also report that the mutant gim1-1 allele behaves as a dominant negative mutation. Introducing t he gim1-1 allele extrachromasomally led to mislocalization of a glycosomal reporter protein even in wild-type cells. Gene disruption experiments in he terozygous GIM1/gim1-1 cells showed that when the mutant gim1-1 allele was lost, cells re-established normal glycosomal protein localization. Interest ingly, no disruptions of the wild-type allele were obtained. These data ind icate that a dominant negative mutation in the GIM1 gene is the sole geneti c lesion responsible for the glycosomal defects in gim1-1, and suggest that GIM1 is an essential gene in Leishmania. (C) 1999 Elsevier Science B.V. Al l rights reserved.