Biology subjects

Landfear, S. M.

Publications and source records attributed to Landfear, S. M..

4 recordsLinked to original sources

bioRxiv2026

Transition Fibers Play a Central Role in Import of a Glucose Transporter into the Flagellar Membrane

Leishmania parasite promastigotes have a motile flagellum that encompasses a variety of integral membrane proteins, but how such proteins traffic selectively to the flagellum is largely unknown. The flagellar-specific glucose transporter, GT1, binds to the KHARON protein via the transporter hydrophilic amino terminal domain, and KHARON is essential for trafficking of GT1 from the flagellar pocket membrane at the base of the flagellum into the flagellar membrane. A major objective of this study was to determine the mechanism whereby KHARON transports GT1 into the flagellar membrane. We have determined that KHARON is a novel component of the transition fibers that project from the basal body at the base of the flagellum toward the flagellar membrane. We propose that KHARON binds GT1 at the transition fibers and then transfers it to intraflagellar transport (IFT) trains as they enter the flagellum at this site. The IFT trains, associated with the membrane tethering protein TULP, then move GT1 through the transition zone filter into the body of the flagellum. These results provide evidence for a role for transition fibers in transport of membrane proteins into flagella.

cell biology↗

bioRxiv2020

Distinct cis-acting elements govern purine-responsive regulation of the Leishmania donovani nucleoside transporters, LdNT1 and LdNT2

Purine salvage from the host is an obligatory process for all protozoan parasites. In Leishmania donovani, this is accomplished by four membrane nucleoside and nucleobase transporters, or LdNTs. Previously, we demonstrated that purine starvation invokes a robust stress response in Leishmania and characterized the proteomic changes involved. However, because Leishmania do not control the transcription of individual genes, the underlying mechanisms responsible for these changes were ill-defined. LdNT1 and LdNT2 are among the most rapidly and significantly upregulated genes in purine-starved L. donovani parasites. Thus, to better understand post-transcriptional mechanisms of purine-responsive gene expression, we have examined regulation of these genes in molecular detail. Here we report that LdNT1 and LdNT2 are controlled by distinct cis-acting elements. In the case of LdNT2, mRNA abundance and translational enhancement under purine stress depend on a 76 nt-long polypyrimidine tract encoded in the LdNT2 mRNA 3-UTR. Transcripts containing the LdNT2 polypyrimidine tract were found to localize to discrete cytoplasmic foci in purine-replete cells, suggesting that the LdNT2 message may be stored in RNA granules at steady-state. In the case of LdNT1, we found that purine-responsiveness is conferred by a 48 nt-long polypyrimidine tract and additional upstream element, termed UE1. Both features are independently required for regulation, with the polypyrimidine tract and UE1 controlling mRNA abundance and translation, respectively. Together, these results highlight a remarkable degree of complexity in the regulation of the Leishmania purine stress response and set the stage for future investigations to identify the larger network of RNA-protein and protein-protein interactions involved.

genetics↗

bioRxiv2020

The Trypanosoma brucei Cytoskeletal Protein KHARON Associates with Partner Proteins to Mediate Both Cytokinesis and Trafficking of Flagellar Membrane Proteins

In the African trypanosome Trypanosoma brucei, the cytoskeletal protein TbKHARON is required for trafficking of a putative Ca2+ channel to the flagellar membrane, and it is essential for parasite viability in both the mammalian stage bloodstream forms and the tsetse fly procyclic forms. This protein is located at the base of the flagellum, in the pellicular cytoskeleton, and in the mitotic spindle in both life cycle forms, and it likely serves multiple functions for these parasites. To begin to deconvolve the functions of KHARON, we have investigated partners associated with this protein and their roles in parasite biology. One KHARON associated protein, TbKHAP1, is a close interaction partner that can be crosslinked to KHARON by formaldehyde and pulled down in a molecular complex, and it colocalizes with TbKHARON in the basal body at the base of the flagellum. Knockdown of TbKHAP1 mRNA has similar phenotypes to knockdown of its partner TbKHARON, impairing trafficking of the Ca2+ channel to the flagellar membrane and blocking cytokinesis, implying that the TbKHARON/TbKHAP1 complex mediates trafficking of flagellar membrane proteins. Two other KHAPs, TbKHAP2 and TbKHAP3, are in close proximity to TbKHARON, but knockdown of their mRNAs does not affect trafficking of the Ca2+ channel. Two different flagellar membrane proteins, which are extruded from the flagellar membrane into extracellular vesicles, are also dependent upon TbKHARON for flagellar trafficking. These studies confirm that TbKHARON acts in complexes with other proteins to carry out various biological functions, and that some partners are involved in the core activity of targeting membrane proteins to the flagellum.

cell biology↗

bioRxiv2020

A Cytoskeletal Protein Complex is Essential for Division of Intracellular Amastigotes of Leishmania mexicana

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, that associate with KHARON. KHAP1 is located only in the subpellicular microtubules, while KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both the KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

microbiology↗