November 28
PROGRAM

02

Glycoscience : diversity and integration

Intracellular trafficking of ceramide bythe ceramide trafficking protein CERTCERT

Director of Department of Biochemistry and Cell Biology,
National Institute of Infectious Diseases, JAPAN22

the 20th Anniversary of the Mizutani Foundation for Glycoscienceno globular domains, but has the short peptide FFAT motif to interact with VAP, an ER
membrane protein. Mutations in the FFAT
motif of CERT impair not only the VAP
interaction but also ER-to-Golgi traffickingof ceramide, although the FFAT mutations
do not affect the activity of CERT to catalyze ceramide transfer between artificial
phospholipid vesicles 4). The middle region
receives phosphorylations that regulate
the function of CERT 5). On the basis ofthese results, we have proposed that CERT
extracts ceramide from the ER and carries
it to the Golgi apparatus in a non-vesicularmanner and that efficient CERT-mediatedtrafficking of ceramide occurs at membrane contact sites between the ER and theGolgi apparatus (Figure.2) 4)6).
In addition, we had previously developed
the chemical inhibitor HPA12 of intracellular trafficking of ceramide, and foundthat the HPA compound is an antagonist ofCERT 7)8). The CERT START domain has apair of tryptophan residues likely responsible for interaction with phospholipidmembranes 3)9). Comparison of co-crystals
revealed that two side chains of the tryptophan pair are protruded outwards in thecomplex with ceramide, but not with theinhibitor, suggesting an unexpected modeof inhibition mechanism by HPA series10).

FFAT motif

START
598 aa, 68 kD

Functions

PH# Golgi-targeting

MR# Interaction with the ER via a FFAT motif# Regulation of CERT function by phosphorylation# Unknown other functions?

START# Inter-membrane transfer of ceramide

Kentaro Hanada

Synthesis and sorting of lipids are essential
events for membrane biogenesis. Intracellular trafficking of lipids may also be important for lipid-mediated signaling events.
Ceramide is synthesized at the endoplasmic reticulum (ER), and transported to
the Golgi compartment for conversion tosphingomyelin (SM). We have identified a
68-kDa cytosolic protein named CERT to
be a key factor for ER-to-Golgi traffickingof ceramide 1). CERT consists of three parts(Figure.1). The amino terminal ~120 aminoacid region forms a pleckstrin homology(PH) domain, which has a Golgi-targeting
function by recognizing phosphatidylinositol-4-monophosphate (PI4P). The carboxyl terminal ~230 amino acid region
forms a START domain capable of extracting ceramide from membranes and transferring the bound ceramide to membranes.
CERT can transfer various molecular species of ceramide but not other lipid types
including sphingosine and SM, indicating its substrate specificity to ceramide
and also flexibility to natural isoforms ofceramide 1)2). X-ray structural analysis ofco-crystals of the CERT START domain incomplex with ceramide has revealed how
CERT exhibits the substrate specificity andflexibility 3). The middle region between thePH and START domains appears to form

Middle region★

Multiple
G67E mutation
phosphorylation
in LY-A cells
sites

Kentaro Hanada, PhD

ProfileKentaro Hanada received his Bachelor degree of Pharmaceutical Science from The
University of Tokyo in 1983, and Ph.D. degree (supervisor: Prof. Yasuhiro Anraku)
from the same university in 1988. During
undergraduate period, he succeeded in purification and reconstitution of an aminoacid transporter from bacteria, which isprobably the first instance for functional
purification of membrane proteins withmultiple TMD by using a biotechnologically linked ‘tag’. In 1988, he joined Department of Chemistry, National Instituteof Health of Japan (afterward renamedto Department of Biochemistry and Cell
Biology, National Institute of Infectious
Diseases) as a research staff and started
biochemical and genetic studies on sphingolipid metabolism with Drs. Yuzuru Akamatsu and Masahiro Nishijima. He isolated a CHO cell mutant defective in serine
palmitoyltransferase, the enzyme for thefirst step of the sphingolipid biosynthesis,
and showed that sphingolipids are essential for the growth of mammalian cells.
From 1993 to 1995, he stayed in the late Dr.
Richard Pagano’s laboratory in USA withLong-term Fellowship of Human Frontier
Science Program and worked on isolationof CHO cell mutant defective in uptake ofa fluorescent phosphatidylserine analog.
After return to Japan, he attempted to isolate various types of CHO cell mutants forsphingomyelin metabolism and found a
new mutant with dysfunction of ceramide
trafficking. Then, he identified CERT, themolecular machinery for ER-to-Golgi
transport of ceramide, and opened a way tounderstand molecular mechanisms underlying intracellular sorting of lipids. Currently, he is the Director of Department ofBiochemistry and Cell Biology, NIID.

PH

Figure 1 Structure of CERT

ER〜10 nm

VAP

CERT

trans Golgi“Short distance shuttle”
Ceramide“Neck swing”
SM

Pl4P

Figure 2. P ossible models for CERT-mediated trafficking of ceramide at the ER-Golgi membrane contact sites

References1) 	Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M, Nishijima M. Molecular machinery for non-vesicular trafficking of ceramide. Nature (246) : 803-809, 2003.
2) 	Kumagai K, Yasuda S, Okemoto K, Nishijima M, Kobayashi S, Hanada K.
CERT mediates intermembrane transfer of various molecular species of ceramides. J. Biol. Chem. (280): 6488-6495, 2005.
3) 	Kudo N, Kumagai K, Tomishige N, Yamaji T, Wakatsuki S, Nishijima M,
Hanada M, Kato R. Structural basis for specific lipid recognition by CERT
responsible for nonvesicular trafficking of ceramide. Proc. Natl. Acad. Sci.
USA (105) 488-493, 2008.
4) 	Kawano M, Kumagai K, Nishijima M, Hanada K. Efficient trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus requires aVAMP-associated protein-interacting FFAT motif of CERT. J. Biol. Chem.
(281) 30279-30288, 2006.
5) 	Kumagai K, Kawano M, Shinkai-Ouchi F, Nishijima M, Hanada K. Interorganelle trafficking of ceramide is regulated by phosphorylation-dependent
co-operativity between the PH and START domains of CERT. J. Biol. Chem.

(282): 17758-17766, 2007.
6) 	Hanada K. Kumagai K, , Tomishige N, Yamaji T. CERT-mediated traffickingof ceramide. Biochim. Biophys. Acta (1791): 684-691, 20097) 	Yasuda S, Kitagawa H, Ueno M, Ishitani H, Fukasawa M, Nishijima M, Kobayashi S, Hanada K. A novel inhibitor of ceramide trafficking from endoplasmic reticulum to the site of sphingomyelin synthesis. J. Biol. Chem. (276):43994-44002, 20018) 	Kumagai K, Yasuda S, Okemoto K, Nishijima M, Kobayashi S, Hanada K.
CERT mediates intermembrane transfer of various molecular species of ceramides. J. Biol. Chem. (280): 6488-6495, 20059) 	Sugiki T, Takahashi H, Nagasu M, Hanada K, Shimada I. Real-time assay
method of lipid extraction activity. Anal. Biochem. (399): 162-167, 201010) 	Kudo N, Kumagai K, Matsubara R, Kobayashi S, Hanada K, Wakatsuki S,Kato R. Crystal structures of the CERT START domain with inhibitors provide insights into the mechanism of ceramide transfer. J. Mol. Biol. (396) :245-251, 2010

Commemorative Symposium

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