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Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs.

ABSTRACT: Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.

SUBMITTER: Abe M

PROVIDER: S-EPMC6527166 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs.

Abe Michiko M Saito Mayu M Tsukahara Ayana A Shiokawa Shuka S Ueno Kazuma K Shimamura Hiroki H Nagano Makoto M Toshima Junko Y JY Toshima Jiro J

The Journal of biological chemistry 20190405 20

Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined ...[more]

PMID: 30952699

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Project description:Despite over a billion years of evolutionary divergence, several thousand human genes possess clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in one or both lineages. These duplicated genes may have been free to diverge in function since their expansion, and it is unclear how or at what rate ancestral functions are retained or partitioned among co-orthologs between species and within gene families. Thus, in order to investigate how ancestral functions are retained or lost post-duplication, we systematically replaced hundreds of essential yeast genes with their human orthologs from gene families that have undergone lineage-specific duplications, including those with single duplications (1 yeast gene to 2 human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a variable pattern of replaceability across different ortholog classes, with an obvious trend toward differential replaceability inside gene families, and rarely observe replaceability by all members of a family. We quantify the ability of various properties of the orthologs to predict replaceability, showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and tissue-specific expression of the human co-orthologs, i.e., the human proteins that are less diverged from their yeast counterpart and more ubiquitously expressed across human tissues more often replace their single yeast ortholog. These trends were consistent with in silico simulations demonstrating that when only one ortholog can replace its corresponding yeast equivalent, it tends to be the least diverged of the pair. Replaceability of yeast genes having more than 2 human co-orthologs was marked by retention of orthologous interactions in functional or protein networks as well as by more ancestral subcellular localization. Overall, we performed >400 human gene replaceability assays, revealing 50 new human-yeast complementation pairs, thus opening up avenues to further functionally characterize these human genes in a simplified organismal context.

Project description:BackgroundMultiprotein-bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP) like proteins (e.g. Gcn4 in yeast) or steroid/nuclear-hormone receptor family (e.g. FTZ-F1 in insect)) and the TATA-box binding protein (TBP) in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Δ) as well as domain swap experiments between aMBF1 and yMbf1 were performed.ResultsIn contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human) the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Δ). Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1.However, as reported previously for Bombyx mori, the C-terminal part of yeast Mbf1 was shown to be not essential for function. In addition phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence, except of two of the three Thaumarchaeota; Cenarchaeum symbiosum A and Nitrosopumilus maritimus SCM1.ConclusionsThe absence of MBF1-interacting activators in the archaeal domain, the presence of a Zn ribbon motif in the divergent N-terminal domain of aMBF1 and the complementation experiments using archaeal- yeast chimeric proteins presented here suggests that archaeal MBF1 is not able to functionally interact with the transcription machinery and/or Gcn4 of S. cerevisiae. Based on modeling and structural prediction it is tempting to speculate that aMBF1 might act as a single regulator or non-essential transcription factor, which directly interacts with DNA via the positive charged linker or the basal transcription machinery via its Zn ribbon motif and the HTH domain. However, also alternative functions in ribosome biosynthesis and/or functionality have been discussed and therefore further experiments are required to unravel the function of MBF1 in Archaea.

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Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs.

Publications

Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs.

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