Deciphering the role of UBA-like domains in intraflagellar distribution and functions of myosin XXI in Leishmania.

Friedrich Frischknecht,Rani Bajaj,Chhitar M Gupta,Bindu Ambaru

doi:10.1371/journal.pone.0232116

Friedrich Frischknecht, Rani Bajaj + Show 2 more

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https://doi.org/10.1371/journal.pone.0232116

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Abstract

Myosin XXI (Myo21) is a novel class of myosin present in all kinetoplastid parasites, such as Trypanosoma and Leishmania. This protein in Leishmania promastigotes is predominantly localized to the proximal region of the flagellum, and is involved in the flagellum assembly, cell motility and intracellular vesicle transport. As Myo21 contains two ubiquitin associated (UBA)-like domains (UBLD) in its amino acid sequence, we considered it of interest to analyze the role of these domains in the intracellular distribution and functions of this protein in Leishmania cells. In this context, we created green fluorescent protein (GFP)-conjugates of Myo21 constructs lacking one of the two UBLDs at a time or both the UBLDs as well as GFP-conjugates of only the two UBLDs and Myo21 tail lacking the two UBLDs and separately expressed them in the Leishmania cells. Our results show that unlike Myo21-GFP, Myo21-GFP constructs lacking either one or both the UBLDs failed to concentrate and co-distribute with actin in the proximal region of the flagellum. Nevertheless, the GFP conjugate of only the two UBLDs was found to predominantly localize to the flagellum base. Additionally, the cells that expressed only one or both the UBLDs-deleted Myo21-GFP constructs possessed shorter flagellum and displayed slower motility, compared to Myo21-GFP expressing cells. Further, the intracellular vesicle transport and cell growth were severely impaired in the cells that expressed both the UBLDs deleted Myo21-GFP construct, but in contrast, virtually no effect was observed on the intracellular vesicle transport and growth in the cells that expressed single UBLD deleted mutant proteins. Moreover, the observed slower growth of both the UBLDs-deleted Myo21-GFP expressing cells was primarily due to delayed G2/M phase caused by aberrant nuclear and daughter cell segregation during their cell division process. These results taken together clearly reveal that the presence of UBLDs in Myo21 are essentially required for its predominant localization to the flagellum base, and perhaps also in its involvement in the flagellum assembly and cell division. Possible role of UBLDs in involvement of Myo21 during Leishmania flagellum assembly and cell cycle is discussed.

Highlights

Myosins are a diverse group of actin-based motor proteins, which are involved in several cellular activities, such as cell motility, intracellular trafficking, cytokinesis, muscle contraction, etc. [1]
Our results indicate that-(I) both the ubiquitin associated (UBA)-like domains are essentially required for prominent localization of Myo21 to the proximal region of the flagellum and flagellum assembly, and (II) only one of the two UBA-like domains may perhaps be sufficient to maintain the normal activity of Myo21 during intracellular vesicle transport and cell division
UBA-like domains, in general, have been reported to be about 35–45 amino acid residues long, and are present in proteins that are involved in cell cycle regulation, DNA repair and ubiquitin/proteasome pathways [15]

Summary

Introduction

Myosins are a diverse group of actin-based motor proteins, which are involved in several cellular activities, such as cell motility, intracellular trafficking, cytokinesis, muscle contraction, etc. [1]. These proteins are composed of a heavy chain, which consists of N-terminal head domain (motor domain) that contain actin and ATP binding sites and possesses ATPase activity, a light chain (calmodulin)-binding neck domain, and a C-terminal tail domain that imparts functional specificity to different classes of myosins [2]. Kinetoplastid parasites such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania all contain one class I myosin and one myosin belonging to a novel class of myosins, class XXI. In addition to these two myosins, T. cruzi contains additional six more myosins, five of which fall into class XXI, whereas TcMyo still remains unclassified [5,6]. The tail region of kinetoplastid myosins contains protein domains that were not known earlier to be associated with myosins, e.g., FYVE, WW, or UBA (Ubiquitin associated)-like domains [5]

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