Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain.

Abstract

Abstract. Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH~-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence, These experiments demonstrate that iso-1, 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 is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain.F IACILITATED glucose transporters are integral membrane proteins (31), present in a diverse spectrum of organisms from bacteria to humans, that shuttle glucose across the plasma membrane and allow uptake and subsequent metabolism of this vital nutrient. Many organisms possess multiple glucose transporter isoforms with distinct biochemical properties (35), and some of these isoforms are localized to different subcellular compartments. Thus, mammalian GLUT1 is targeted primarily to the plasma membrane in fat and muscle cells, whereas GLUT4 is directed primarily to intracellular vesicles (32) that fuse with the plasma membrane after insulin stimulation of the target cell. A problem of considerable interest has been to determine how such isoforms are differentially targeted to their respective subcellular locations. Previous studies on the parasitic protozoan Leishmania enriettii have revealed that this microorganism expresses two closely related isoforms (34) of a major glucose transporter designated Pro-1. Both isoforms of the Pro-1 transporter are expressed in the promastigote or insect stage of the parasite life cycle, but their mRNAs accumulate to only a residual level in the amastigote form of the parasite that lives within the macrophage lysosomes of the vertebrate host (4). These two isoforms (iso-1 and iso-2) possess distinct NH2-terminal hydrophilic domains, predicted to be orien...

SMP-1, a Member of a New Family of Small Myristoylated Proteins in Kinetoplastid Parasites, Is Targeted to the Flagellum Membrane inLeishmania

“…A number of trypanosomatid proteins appear to be localized to the plasma membrane of the cell body through interactions with the underlying microtubule cytoskeleton (Piper et al, 1995;Hertz-Fowler et al, 2001;Vedrenne et al, 2002). However, several lines of evidence suggest that the flagellar localization of SMP-1 is not dependent on interactions with the axoneme microtubules or the paraflagellar rod.…”

“…Studies on trypanosomatid flagellar membrane proteins have provided new insights into the signals that target proteins to the flagellar membrane. Specifically, sorting of the L. enrietti glucose transporter isoform, ISO1, to detergent-soluble membranes in the flagellum, is mediated by a peptide sequence within the N-terminal cytoplasmic tail of this polytopic membrane protein (Piper et al, 1995;Snapp and Landfear, 1999). In contrast, dual acylation of the amino termini Article published online ahead of print.…”

Secretory Pathway of Trypanosomatid Parasites

“…Glucose transporter isoform 2 is directed to the cell body by a short N-terminal cytoplasmic tail that mediates the attachment of this protein to the subpellicular microtubules that underlie the cell body (331). On the other hand, glucose transporter isoform 1 has a longer (139-amino-acid) cytoplasmic domain that contains a flagellarmembrane-targeting motif (266,330,331). From alanine-scanning mutagenesis, this targeting motif was restricted to five contiguous amino acids.…”

Nutrient Transport and Pathogenesis in Selected Parasitic Protozoa

“…The GT2 and GT3 proteins are present in the pellicular plasma membrane (PPM) that surrounds the cell body, but they are not present at detectable levels in the flagellar membrane. In contrast, GT1 is localized primarily to the flagellar membrane (81). The function of the flagellar localization of GT1 is still not clear, but two sequences within the unique amino-terminal hydrophilic region of this permease have been implicated in flagellar targeting (70).…”