Abstract
Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes.
Highlights
Several metabolic enzymes have been identified to be able to form visible intracellular structures in a variety of species, from bacteria to yeast, fly, and even mammals [1,2,3,4,5,6,7,8,9,10]
To help yeast asparagine synthetases move across the nuclear membrane, we decided to introduce the coding sequence of green fluorescent protein (GFP; to visualize the localization of the fluorescently tagged protein under fluorescence microscope) and the coding sequence of nuclear localization signal (NLS; to make the fusion protein capable of nuclear transport) [30] downstream of the coding sequence of ASN1, in the yeast genome (Fig 1)
This approach can ensure that the expression of GFP-NLS-tagged asparagine synthetases
Summary
Several metabolic enzymes have been identified to be able to form visible intracellular structures in a variety of species, from bacteria to yeast, fly, and even mammals [1,2,3,4,5,6,7,8,9,10]. One of the most studied metabolic enzymes possessing self-assembly is CTP synthetase, the enzyme responsible for making CTP which is a key precursor for nucleic acid and lipid metabolism [11, 12]. Innovation (MHESI), (3) the Development and Promotion of Science and Technology Talents Project (DPST), and (4) Mahidol University (MU)
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