Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae.
Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.
Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand.
Anja
A
Wilmes
Reem
R
Hanna
Rosemary W
RW
Heathcott
Peter T
PT
Northcote
Paul H
PH
Atkinson
David S
DS
Bellows
John H
JH
Miller
D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects* / Q000235:genetics*; D006720:Homozygote; D007783:Lactones / Q000494:pharmacology*; D064246:Mad2 Proteins; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D008941:Spindle Apparatus / Q000187:drug effects / Q000235:genetics; D014404:Tubulin / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; D018797:Cell Cycle Proteins; D007783:Lactones; C577695:MAD2 protein, S cerevisiae; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; C414085:peloruside A; D017239:Paclitaxel