KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes

Abstract

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.

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

“…Previous studies on the L. mexicana flagellar glucose transporter GT1 established that a cytoskeletal protein, designated KH, interacts with the flagellar targeting motif of that permease and is involved in trafficking GT1 to the flagellar membrane (4). KH in both L. mexicana and T. brucei is distributed over microtubule structures at the base of the flagellum, the pellicular cytoskeleton, and the mitotic spindle (4,6). Furthermore, studies with either gene knockouts in L. mexicana (4,5) or RNAi in T. brucei (6) established that KH is required for trafficking of some proteins to the flagellar membrane, for viability of infectious stage parasites, and for cytokinesis.…”

“…However, flagella or their component cytoskeletons can be isolated from T. brucei with the kinetoplast DNA, basal body, and proximal regions intact (37)(38)(39). Hence, that parasite offers a potentially tractable model for identifying KH Complex 1 components, and orthologs in both T. brucei and L. mexicana could be tested for roles in transport of flagellar membrane proteins such as TbCaCh/FS179, a putative Ca 2+ channel in T. brucei that is dependent upon TbKHARON for flagellar localization (6), and GT1 (4), respectively.…”

“…Because one of the three locations observed for KH was the mitotic spindle (6), it was also important to determine whether KHAP1 or KHAP2 associated with KHARON at this site. Promastigotes expressing KHAP1::mNG were stained with the anti-b-tubulin KMX-1 antibody that allows visualization of mitotic spindles (22), and parasites in the process of division were imaged.…”

“…The intracellular amastigotes were able to replicate nuclei, but parasites could not undergo cytokinesis, leading to multinucleate, multiflagellated amastigotes that eventually disintegrated and were thus unable to support a productive infection (5). Localization studies indicated that KH is a cytoskeletal-associated protein and is attached to i) microtubule-based structures at the base of the flagellum (4), ii) the network of subpellicular microtubules that subtend the cytosolic side of the plasma membrane throughout the parasite cell body (4), and iii) mitotic spindles (6).…”

A Novel Calcium-Activated Potassium Channel Controls Membrane Potential and Intracellular pH in Trypanosoma cruzi

“…In contrast, TcCAKC expression and analysis by two-electrode voltage clamp produced robust and reproducible currents that require activation by calcium, indicating that TcCAKC encodes an α-pore forming subunit able to traffic and assemble into functional channels. No accessory β-subunits of K + channels have been identified in T. cruzi, but recent reports have shown that KHARON, a protein complex unique to trypanosomatids, is required for correct targeting of calcium in T. brucei (Sanchez et al, 2016) and glucose transporters in Leishmania (Tran et al, 2013), highlighting a non-canonical pathway for membrane protein trafficking in these parasites.…”