Molecular Cloning and Characterization of a Candidate ENOX Protein of Saccharomyces cerevisiae with a 25 Min Period Insensitive to Simalikalactone D Inhibition and Melatonin
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Author(s)
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Department of Biology, Valparaiso University, Valparaiso, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Department of Biology, Valparaiso University, Valparaiso, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
Mor-NuCo, Inc., Purdue Research Park, West Lafayette, USA.
A yeast deletion library was screened based on NADH
fluorescence using a 384-well plate assay to identify a yeast isolate
lacking a previously identified cell surface oxidase exhibiting an
oscillatory pattern with a period length of 25 min and resistant to the
ENOX1-specific inhibitor simalikalactone D (YNOX for yeast-specific ENOX
= ENOX4). The cDNA was cloned from a yeast over expression library
using NADH fluorescence analyzed by Fast Fourier transform and
decomposition fits. The objective was to identify and sequence an ENOX
homologue in Saccharomyces cerevisiae with a 25 min rather than a 24 min
period length (YNOX). The finding identified YDR005C as the yeast ENOX
protein with a temperature-independent 25 min period length and
insensitive to inhibition by simalikalactone D. The encoded protein was
expressed in bacteria and characterized. Gel slices corresponding to 55
kDa and 39 kDa His-tagged proteins exhibited 25 min oscillatory patterns
not inhibited by 1 μM simalikalactone D for both NADH oxidation and
reduced coenzyme Q10 oxidation as well as a protein
disulfide-thiol interchange activity which alternated with the oxidative
activities. Activities were phased by low-frequency electromagnetic
fields but, in contrast, to yeast ENOX1, not by addition of melatonin.
The assay in the presence of D2O shifted the length of the
oscillatory period from 25 min to 32 min. The YDR005C deletion mutant
cells lacked the ENOX4 clock output present in the wild type yeast.
KEYWORDS
Cite this paper
Ryuzoji, A. , Parisi, D. , Dick, S. , Kim, J. ,
Morré, D. and Morré, D. (2014) Molecular Cloning and Characterization
of a Candidate ENOX Protein of Saccharomyces cerevisiae with a 25 Min Period Insensitive to Simalikalactone D Inhibition and Melatonin. Advances in Biological Chemistry, 4, 339-350. doi: 10.4236/abc.2014.45039.
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