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The conserved hydrophobic sequence LLI of yeast Spo7 is required for its regulatory role in Nem1‐Spo7 phosphatase function
Author(s) -
Mirheydari Mona,
Han GilSoo,
Carman George M
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.488.11
Subject(s) - phosphatidate , dephosphorylation , biochemistry , phosphatase , mutagenesis , biology , function (biology) , diacylglycerol kinase , mutant , microbiology and biotechnology , phosphorylation , gene , protein kinase c
Nem1 (catalytic subunit) and Spo7 (regulatory subunit) form a nuclear/endoplasmic reticulum‐associated protein phosphatase complex that regulates the phosphorylation state and function of phosphatidate phosphatase Pah1 in yeast. By dephosphorylating Pah1, the Nem1‐Spo7 phosphatase facilitates the association of Pah1 with the membrane for its function to convert phosphatidate to diacylglycerol for triacylglycerol synthesis. In this work, a mutagenesis analysis of Spo7 was conducted to gain information on the structural requirements for the protein's role in regulating Nem1‐Spo7 phosphatase function. A nested deletion analysis of the N‐terminal region of Spo7 indicated that amino acid residues 46–56 were required for Nem1‐Spo7 function as scored by a temperature sensitivity assay of yeast cells common to that exhibited by cells lacking Pah1. A conserved hydrophobic sequence LLI is contained within this region, and the importance of this sequence to Spo7 function was examined by site‐directed mutagenesis. Whereas all three hydrophobic amino acids affected Nem1‐Spo7 function, Leu‐54 had the greatest effects on lipid metabolism. Cells carrying the L54R mutation had much reduced levels of triacylglycerol and elevated levels of phospholipids and fatty acids, indicating a defect in the dephosphorylation and function of Pah1 phosphatidate phosphatase activity. The importance of Leu‐54 to the interactions of Spo7 with Nem1 and/or Pah1 are currently under investigation. Support or Funding Information Supported by NIH grant GM050679 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .