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Creation of a myosin Va-TAP-tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum.

ABSTRACT: The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.

SUBMITTER: Alexander CJ

PROVIDER: S-EPMC8365777 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Metabolic pathways are largely conserved in eukaryotes, but the transcriptional regulation of these pathways can sometimes vary between species; this has been termed rewiring. Recently it has been established that in the Saccharomyces lineage starting from Saccharomyces castelli, genes involved in allantoin breakdown have been genomically relocated to form the DAL cluster. The formation of the DAL cluster occurred along with the loss of urate permease (UAP) and urate oxidase (UOX), reducing the requirement for oxygen and bypassing the candidate Ppr1 inducer, uric acid. In Saccharomyces cerevisiae this allantoin catabolism cluster is regulated by the transcription factor Dal82, which is not present in many of the pre-rearrangement fungal species. We have used ChIP-Chip analysis, transcriptional profiling of an activated Ppr1 protein, bioinformatics, and nitrogen utilization studies, to establish that in Candida albicans the zinc cluster transcription factor Ppr1 controls this allantoin catabolism regulon. Intriguingly, in S. cerevisiae the Ppr1 ortholog binds the same DNA motif (CGG(N6)CCG) as in C. albicans, but serves as a regulator of pyrimidine biosynthesis. This transcription factor rewiring appears to have taken place at the same phylogenetic step as the formation of the rearranged DAL cluster. This transfer of the control of allantoin degradation from Ppr1 to Dal82, together with the repositioning of Ppr1 to the regulation of pyrimidine biosynthesis, may have resulted from a switch to a metabolism that could exploit hypoxic conditions in the lineage leading to S. castellii and S. cerevisiae. Metabolic pathways are largely conserved in eukaryotes, but the transcriptional regulation of these pathways can sometimes vary between species; this has been termed rewiring. Recently it has been established that in the Saccharomyces lineage starting from Saccharomyces castelli, genes involved in allantoin breakdown have been genomically relocated to form the DAL cluster. The formation of the DAL cluster occurred along with the loss of urate permease (UAP) and urate oxidase (UOX), reducing the requirement for oxygen and bypassing the candidate Ppr1 inducer, uric acid. In Saccharomyces cerevisiae this allantoin catabolism cluster is regulated by the transcription factor Dal82, which is not present in many of the pre-rearrangement fungal species. We have used ChIP-Chip analysis, transcriptional profiling of an activated Ppr1 protein, bioinformatics, and nitrogen utilization studies, to establish that in Candida albicans the zinc cluster transcription factor Ppr1 controls this allantoin catabolism regulon. Intriguingly, in S. cerevisiae the Ppr1 ortholog binds the same DNA motif (CGG(N6)CCG) as in C. albicans, but serves as a regulator of pyrimidine biosynthesis. This transcription factor rewiring appears to have taken place at the same phylogenetic step as the formation of the rearranged DAL cluster. This transfer of the control of allantoin degradation from Ppr1 to Dal82, together with the repositioning of Ppr1 to the regulation of pyrimidine biosynthesis, may have resulted from a switch to a metabolism that could exploit hypoxic conditions in the lineage leading to S. castellii and S. cerevisiae.

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Creation of a myosin Va-TAP-tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum.

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