DIFFERENTIAL TARGETING OF 2 GLUCOSE TRANSPORTERS FROM LEISHMANIA-ENRIETTII IS MEDIATED BY AN NH2-TERMINAL DOMAIN

Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect ve ctor and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. e nriettii exists as two isoforms, iso-l and iso-2, which are both expre ssed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and ex tensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have l ocalized these glucose transporters within promastigotes by two approa ches. In the first method, we have raised a polyclonal antibody agains t the COOH-terminal hydrophilic domain shared by both iso-l and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The stai ning observed with this antibody occurs primarily on the plasma membra ne and the membrane of the flagellar pocket, but there is also light s taining on the flagellum. We have also localized each isoform separate ly by introducing an epitope tag into each protein sequence. These exp eriments demonstrate that iso-l, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform i s differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides withi n the NH2-terminal hydrophilic domain.