| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Research Abstract |
CERT is a cytoplasmic protein that mediates ceramide-specific intracellular transport from endoplasmic reticulum (ER) to Golgi compartment. CERT contains two lipid-binding domains, PH and START domains. The N-terminal PH domain (CERT PH) interacts specifically with phosphatidylir, ositol 4-monophosphate (Ptdlns(4)P). The C-terminal START domain catalyzes ceramide transfer. CERT is targeted on the Golgi membrane which expresses abundant Ptdlns(4)P. This targeting enables CERT to transfer ceramide from ER to Golgi. A mutant G67E on PH domain completely losses ceramide transports activity. Therefore, the interaction between CERT PH and Golgi-membrane is crucial for ceramide transport activity of CERT. In the present study, we identified interface on CERT PH with Golgi-membrane mimetic liposome using Transferred cross-saturation (TCS) experiments, an NMR method to identify interaction surface with huge molecules developed in our laboratory.
First, to investigate the recognition mechanism of
… More
Ptdlns(4)P by CERT PH, we performed chemical shift perturbation experiments using a head moiety analogue, Inositol(1,4)P2, and surface plasmon resonance (SPR) experiments using Golgi-membrane mimetic liposomes. Consequently, CERT PH binds polar head group of Ptdlns(4)P with very low affinity at Kd values of approximately 800 tiM, whereas CERT PH recognizes Golgi-membrane mimetic liposomes with higher affinity at Kd values of 6.56 μM. From these results, we hypothesized that CERT PH needs not only the head group of Ptdlns(4)P but also lipid membrane to interact with Golgi-membrane with sufficient affinity.
Next, we performed TCS experiments to elucidate the residues that interact with Ptdlns(4)P and lipid-membrane on CERT PH. As a result, we found the PtdIls(4)P interaction surface on CERT PH comprised of Lys32, Arg43, Tyr54 and Arg66. Moreover, we identified the lipid membrane interaction surface on the loop between β1 and β2 strands and on the β7 strand comprised of Trp33, lle37, His38, Trp40, Tyr96 and Arg98. To confirm the residues on the lipid membrane interaction surface that play an important role in interaction with Golgi-membrane, point mutations were introduced intothem. As a result of a SPR experiments, it was revealed that the Trp33, 11e37, His38 on lipid membrane interaction surface is essential for interaction with Golgi-membrane. Less
|
