Creation of fluorescent unnatural aminophospholipid synthases

2018 Fiscal Year Final Research Report

• Project/Area Number: 17K19338
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Molecular and Genome biology and related fields
• Research Institution: Yamagata University
• Principal Investigator: Yasushi Tamura 山形大学, 理学部, 准教授 (50631876)
• Project Period (FY): 2017-06-30 – 2019-03-31
• Keywords: リン脂質 / オルガネラ / リン脂質輸送

Outline of Final Research Achievements

In this study, we aimed to obtain phosphatidylserine (PS) synthase PssA mutants, which recognize NBD-serine (fluorescence-labeled serine) instead of serine. So far, we created experimental systems in which PssA is expressed in an IPTG-dependent manner in Escherichia coli cells and expressed on various organelle membranes in yeast cells. Interestingly, when PssA is expressed in the ER, lipid droplet, peroxisome or mitochondrial inner membrane, growth defects of cho1-deleted cells were rescued. These results are important for investigating intracellular phospholipid transport pathways. We are now using these yeast cells for genetic screening.
In addition to the genetic method, we tool an advantage of structural analysis. If we were able to determine the recognition site of PssA for the subtrate serine, we could obtain mutants that recognize NBD-serine by introducing mutations to the sites.

Free Research Field

細胞生物学

Academic Significance and Societal Importance of the Research Achievements

大腸菌のPS合成酵素を真核生物に移植することで，本来不可能であった，真核生物内のPS合成の細胞内局在場所を変化させることが出来るようになった。これにより，PSが小胞体で合成される必然性が初めて検証できるようになった。また様々な細胞内区画で合成されたPSの運命（PS→ホスファチジルエタノールアミンへのミトコンドリア内での変換）をモニターすることで，細胞内の脂質の流れをモニターする実験系の構築に成功した。この実験系により，細胞内の異なるおるがねら膜間をかなり効率的に脂質が移動する様子を様々なおるがねら間で調べることが出来るようになった。