Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites

Abstract

SummaryUpregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii , confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/-and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

Global Analysis of Protein Palmitoylation in African Trypanosomes

“…Like the folate/pteridine transporters, this protein has a CCCG peptide in a region between two of its predicted transmembrane domains. Intriguingly, a homologue of this protein in Leishmania similarly appears to undergo growth stage-dependent redistribution to lysosomal compartments for degradation (6). We therefore propose that these proteins are common substrates for a single PAT whose activity confers growth stage-regulated responses.…”

Flavonoid Dimers as Bivalent Modulators for Pentamidine and Sodium StiboglucanateResistance in Leishmania

“…LeMDR1 is known to be responsible for vinblastine and puromycin resistance in L. enriettii (11,16). When we tried to study the modulating activity of the flavonoid dimers on the vinblastine and puromycin resistance of LeV160, we found that none of the flavonoid dimers have any significant modulating activity ( Table 2).…”

“…We have investigated whether the ABC transporter, LeMDR1, in L. enriettii is the target of the synthetic flavonoid dimers. We have previously demonstrated that LeMDR1 is an ABC transporter that can mediate resistance to vinblastine and puromycin and sensitivity to rhodamine 123 (11,16). Here we studied the modulating effect of the synthetic flavonoid dimers on three L. enriettii cell lines, namely, wild-type Le, LeMDR1 knockout (LeMDR1 Ϫ/Ϫ ), and LeMDR1 overexpressed (LeV160).…”

Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins

“…In addition, a polypeptide of ϳ28 kDa that reacted with anti-GFP antibodies was released via proteolysis from the fusion protein. This proteolytically resistant GFP product, also known as prGFP (8,29), was found only in the soluble fraction, as expected, since only the fusion protein carrying TM domains is expected to reside in the membrane fraction. The level of EP was used to examine the effect of the depletion on SP-containing proteins.…”

“…3A2). Such cleaved GFP (prGFP) was first discovered in the Leishmania MVTlysosome (8,29). These data suggest that in wild type cells, the majority of the protein traverses the ER but is mislocalized and is not membrane associated.…”