title: Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the Deltasnf2 disruptant produces a significant increase in its enzyme activity.
abstract: We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the Deltasnf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the Deltasnf2 disruptant. To analyze the expression of Dga1p in wild type and the Deltasnf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the Deltasnf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the Deltasnf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the Deltasnf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1DeltaN2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1DeltaN2p is activated Dga1p. Dga1DeltaN2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, 305-8566, Japan. kamisaka.y@aist.go.jp
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kamisaka
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Kimura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uemura
authors.forename: Motonari
authors.initials: M
authors.lastname: Shibakami
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D055786:Gene Knockout Techniques; D015151:Immunoblotting; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D013194:Staining and Labeling; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C494880:DGA1 protein, S cerevisiae; D051048:Diacylglycerol O-Acyltransferase; D000251:Adenosine Triphosphatases; C499333:SNF2 protein, S cerevisiae