Both sequence and context are important for flagellar targeting of a glucose transporter

Abstract

SummaryMany of the cilia-and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins.

Determinants of GPI-PLC Localisation to the Flagellum and Access to GPI-Anchored Substrates in Trypanosomes

“…Furthermore, residues 1 to 24 were sufficient for localisation of a GFP reporter to the flagellar membrane and this same reporter was localised to the flagellum in Leishmania amazonensis suggesting a conserved mechanism. The second motif was identified in the flagellar glucose transporter 1 from Leishmania mexicana (LmxGT1; LmxM.36.6300) [57]. LmxGT1 contains 12 membrane-spanning segments located after residue 131; the N-terminal 130 residues are present in the cytoplasm.…”

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

“…This model is supported by (i) the observation that KH1 cross-linked to wild type LmxGT1 but not to the ⌬(84 -100) mutant in which critical flagellar targeting sequence, including the NPM motif, has been deleted; (ii) LmxGT1 failed to enter efficiently into the external FM in ⌬kh1 null mutants and is largely retained in the FP, (iii) LmxGT1 encompassing mutations in the NPM motif is also stalled in the FP (17); and (iv) HA-tagged KH1 is located on the proximal flagellar axoneme within the FP but not on the component of the axoneme that extends beyond the FP.…”

“…The cis-acting flagellar targeting signal of LmxGT1 has been described previously (17). Three critical residues within the N-terminal hydrophilic domain, Asn (18,19) has not identified a similar motif among other eukaryotes, supporting the possibility that novel flagellar targeting machinery may exist in these ancient protozoa.…”

“…Additionally, the aquaglyceroporin channel of Leishmania major (LmjAQP1) is specifically targeted to the flagellar membrane where it is involved in detection of extracellular osmotic gradients and osmotaxis (15). Furthermore, the L. mexicana glucose transporter LmxGT1 is also selectively localized to the flagellar membrane where it may act as a glucose sensor (16,17).…”

The Trypanosoma cruzi Surface, a Nanoscale Patchwork Quilt

“…acylation) for these domains could be one possible explanation. However, there are some evidences that other non-lipidic determinants are involved in flagellar localization of membrane proteins in trypanosomatids [46, 50–53]. Thus, although there is not enough information about how proteins are sorted to a specific place in the plasma membrane, it seems that acylation and protein determinants or the combination of both may be associated with this process.…”