Neurotransmitter Transporter-Like: A Male Germline-specific SLC6 Transporter Required for Drosophila Spermiogenesis

The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl), is expressed only in the male germline. Mobilization of a transposon inserted near the 3′ end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism

Divergent polo box domains underpin the unique kinetoplastid kinetochore

Kinetochores are macromolecular machines that drive eukaryotic chromosome segregation by interacting with centromeric DNA and spindle microtubules. While most eukaryotes possess conventional kinetochore proteins, evolutionarily-distant kinetoplastid species have unconventional kinetochore proteins, composed of at least 19 proteins (KKT1–19). Polo-like kinase (PLK) is not a structural kinetochore component in either system. Here we report the identification of an additional kinetochore protein, KKT20, in Trypanosoma brucei. KKT20 has sequence similarity with KKT2 and KKT3 in the Cys-rich region and all three proteins have weak but significant similarity to the polo box domain of PLK. These divergent polo box domains of KKT2 and KKT20 are sufficient for kinetochore localization in vivo. We propose that the ancestral PLK acquired a Cys-rich region and then underwent gene duplication events to give rise to three structural kinetochore proteins in kinetoplastids

Kinetoplastid kinetochore proteins KKT2 and KKT3 have unique centromere localization domainsation domains

The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3

Divergent polo boxes in KKT2 bind KKT1 to initiate the kinetochore assembly cascade in Trypanosoma brucei

Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in&nbsp;Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 directly interact with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains, which initiate kinetochore assembly in&nbsp;T. brucei.</p