LAX28 is required for the stable assembly of the inner dynein arm f complex, and the tether and tether head complex in Leishmania flagella

Abstract

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins; however, the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm (IDA) f complex (also known as the I1 complex), and the tether and tether head (T/TH) complex are thought to be key regulators of dynein action but, unlike the IDA f complex, T/TH proteins remain poorly characterised. Here, we characterised T/TH-associated proteins in the protist Leishmania mexicana. Proteome analysis of axonemes from null mutants for the CFAP44 T/TH protein showed that they lacked the IDA f protein IC140 and a novel 28-kDa axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, both sharing features with sensory BLUF-domain-containing proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility, albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f and T/TH-associated proteins for axonemal assembly in Leishmania.

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“…The band corresponding to FAP44 with lower molecular mass was likely a degradation product or modified form of this protein. The amount of MOT7 was significantly decreased in a Chlamydomonas fap44 mutant (20), and recent work on a homolog of MOT7 in Leishmania, LAX28, showed that it was linked to f/I1 dynein and the tether complex (17). We could not find FAP44 in the Ciona f/I1 dynein fraction, possibly because of differences in the interactions among the tether, DYBLUP, and dynein motor domain in Chlamydomonas and Ciona.…”

“…2E and 3) (20), which mechanically stabilizes f/I1 dynein in the axoneme (20,21). This differs from findings on a homolog of DYBLUP in Leishmania, LAX28, loss of which from the axoneme causes failure in the assembly of both f/I1 dynein and the tether complex (17). Thus, the dependency of assembly of f/I1 dynein on DYBLUP orthologs varies among organisms.…”

“…Leishmania , a genus of the supergroup Excavata, has a homolog of DYBLUP, LAX28 ( 17 ); however, it appears to lack the BLUF domain and shares low homology with the DYBLUP orthologs of animals and Chlamydomonas MOT7. Among other Excavata, Trypanosoma have a DYBLUP homolog with no BLUF domain ( 17 ), and Euglena show no clear ortholog of DYBLUP (fig. S2C).…”