Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes

Abstract

Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing ~81% of human cytoskeletal genes across 7 gene families for their ability to complement a growth defect induced by inactivation or deletion of the corresponding yeast ortholog. In 5 of 7 families-all but α-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phenotypes of humanized strains with deletion phenotypes of their interaction partners, we identify instances of human genes in the actin and septin families capable of carrying out essential functions, but apparently failing to fully complement the cytoskeletal roles of their yeast orthologs, thus leading to abnormal cell morphologies. Overall, we show that duplicated human cytoskeletal genes appear to have diverged such that only a few human genes within each family are capable of replacing the essential roles of their yeast orthologs. The resulting yeast strains with humanized cytoskeletal components now provide surrogate platforms to characterize human genes in simplified eukaryotic contexts.

Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1

“…The apparent Cdc12 ortholog from the filamentous fungus Aspergillus nidulans AspC was able to complement the inviability of a cdc12Δ mutant only poorly, and when expressed in WT cells, it promoted formation of atypical pseudohyphae rather than normal buds, even though it appeared to localize at the bud neck ( Lindsey et al 2010 ). Recently, the major isoforms of all 13 human septin gene products were tested for their ability to rescue cdc3Δ , cdc10Δ , cdc11Δ , and cdc12Δ mutant cells; and only complementation of cdc10Δ cells was observed ( Garge et al 2020 ). Of the 13 human septins, only four—two from homology Group 1A (SEPT3 and SEPT9) and two from homology Group 1B (SEPT6 or SEPT10)—were able to exhibit a Cdc10-like function in vivo but could not fully replace the yeast subunit ( Garge et al 2020 ).…”

“…Recently, the major isoforms of all 13 human septin gene products were tested for their ability to rescue cdc3Δ , cdc10Δ , cdc11Δ , and cdc12Δ mutant cells; and only complementation of cdc10Δ cells was observed ( Garge et al 2020 ). Of the 13 human septins, only four—two from homology Group 1A (SEPT3 and SEPT9) and two from homology Group 1B (SEPT6 or SEPT10)—were able to exhibit a Cdc10-like function in vivo but could not fully replace the yeast subunit ( Garge et al 2020 ). Phylogenetic analysis suggests that human Group 1A and IB septins may share a common ancestor with S. cerevisiae Cdc10 ( Pan et al 2007 ).…”

Humanized yeast to model human biology, disease and evolution

“…In spite of the cellular and organismal differences between humans and yeast, several recent studies have discovered that many conserved protein-coding human genes can substitute for their yeast equivalents and sustain yeast growth ( Garge et al, 2020 ; Hamza et al, 2015 , 2020 ; Kachroo et al, 2015 ; Laurent et al, 2020 ; Sun et al, 2016 ). These assays uncovered which conserved processes are still interchangeable while also creating reagents to directly test human gene function in a tractable system.…”

“…Functional complementation tests of human genes in yeast are not novel and have been explored extensively in the past ( Balakrishnan et al, 2012 ; Heinicke et al, 2007 ). However, a systems approach to test hundreds of shared human genes for functional replaceability in yeast has only recently been reported ( Garge et al, 2020 ; Hamza et al, 2015 , 2020 ; Kachroo et al, 2015 ; Laurent et al, 2020 ; Sun et al, 2016 ) ( Fig. 4 A).…”

Propagation of Mitochondria-Derived Reactive Oxygen Species within the Dipodascus magnusii Cells

“…It is pertinent to note that yeasts and humans, despite their evolutionary divergence that resulted in dramatic differences in cell and tissue organization, motility, and environment [ 64 ], share well-conserved molecular and cellular mechanisms of eukaryotic cell biology. These include commonalities in their underlying molecular makeup, the universal mechanisms that govern signaling pathways [ 65 , 66 ], protein folding, quality control and degradation, mitochondrial dysfunction, oxidative stress, a secretory pathway, vesicular trafficking [ 67 , 68 , 69 ], and even mechanisms of cell death and survival [ 68 , 70 ]. Moreover, numerous processes and mechanisms, such as cell signaling pathways that regulate metabolism, cell growth and division, organelle functions, cellular homeostasis, stress responses, and mitochondrial dynamics (fission and fusion) [ 71 ], were first identified in yeasts and then shown to be conserved in higher eukaryotes.…”