The Middle Region is Required for Phosphorylation Regulation of Ceramide Transfer Protein

The trafficking of ceramide from endoplasmic reticulum (ER), where it is synthesized, to the Golgi for sphingomyelin synthesis is mediated by the ceramide transfer protein (CERT) in a nonvesicular fashion. CERT is a cytosolic protein containing multiple domains and motifs. The START (steroidogenic acute regulatory protein-related lipid transfer) domain at the C-terminus has the ability to transfer ceramide. The N-terminus contains the pleckstrin homology (PH) domain which is able to recognize phosphatidylinositol-4-monophosphate (PtdIns(4)P) enriched in the Golgi membrane. Following the PH domain, there is a ~30 residue stretch that is rich in serine and threonine residues, aptly named the Serine Rich motif (SR). Phosphorylation of this region at multiple sites inhibits CERT transfer of ceramide. The region following the SR motif is the Middle Region (MR) which contains a predicted coiled-coil region. This coiled-coil region is hypothesized to mediate oligomerization of CERT, yet the role of MR is unknown. In this study, we primarily used biochemical assays to investigate the role of MR in CERT function. Our data shows MR is required for PtdIns(4)P enhancement of CERT activity and functional inhibition in the phosphorylation mimic. To further delineate how MR is involved in CERT function, the predicted coiled-coil region was deleted from both the wild-type (WTFL) and the phosphorylation mimic (10EFL) CERT proteins. While it is well established that 10EFL has greatly diminished activity as compared to the WTFL protein, removal of the coiled-coil region abolishes inhibition of the phosphorylation mimic. Collectively, these data suggest coiled-coil region is critical for phosphorylation mimic inhibition of CERT activity. We compared the affinities for PtdIns(4)P containing liposome of WTFL and 10EFL proteins to gain insight into the role of membrane binding in the ceramide transfer activity. The WTFL has about 6 fold higher affinity for PtdIns(4)P containing liposomes than the 10EFL protein, while the coiled coil deleted proteins bind to liposome only slight weaker (1.5 fold) than the WTFL protein. These results suggest CERT affinity for PtdIns(4)P containing liposome plays a critical role in CERT regulation. Furthermore, they imply that the coiled coil region is critical for mediating the reduced affinity of 10EFL protein for the Golgi membrane.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Cellular and Molecular Biology (UMKC)

Grantor

University of Missouri--Kansas City (UMKC)

Year dc:date.issued

2013

Author dc:creator

• Bye, Nicole Elizabeth

Advisor dc:contributor.advisor

• Yao, Xiaolan

Language dc:language.iso

en_US

Handle dc:identifier.uri

https://hdl.handle.net/10355/47454

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/47454