Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

From the Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2012; 287(40):33706-18. DOI: 10.1074/jbc.M112.367730
Source: PubMed


Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the
process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid
transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic
phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide
by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques.
The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4)P recognition site. An extensive
interaction study using NMR and other biophysical techniques revealed that the basic groove coordinates the CERT PH domain
for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization
of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove
is conserved only in the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with
the signal transduction activity.

9 Reads
  • Source
    • "Ceramide transfer protein (CERT) is responsible for the active transport of majority of the ceramide from ER to the Golgi complex where ceramide is converted to sphingomyelin and other complex sphingolipids [4]. CERT is a cytosolic protein that interacts with both ER and Golgi components to facilitate ceramide transfer [5], [6], [7], [8], [9]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.
    PLoS ONE 03/2014; 9(3):e92142. DOI:10.1371/journal.pone.0092142 · 3.23 Impact Factor
  • Source
    • "The effect of PtdIns(4)P was overridden by increasing the concentration of UDP-glucose in the reaction solution, suggesting that PtdIns(4)P may inhibit contact between the active site of UGCG and UDP-glucose (Fig. 3). The interaction between PtdIns(4)P and the PH domain of CERT is indispensable for the transport of ceramide from ER to Golgi [50]. Therefore, CERT-transported ceramide may colocalize with PtdIns(4)P, thereby inhibiting, or interrupting, transfer of glucose from UDPglucose to ceramide by UGCG (Fig. 3). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids.
    Biochimica et Biophysica Acta 06/2013; 1831(9). DOI:10.1016/j.bbalip.2013.06.001 · 4.66 Impact Factor
  • Source
    • "However, in the presence of phosphatidylserine, it exhibits stronger and more specific interaction over other differently phosphorylated PIPs (Hamard-Peron et al., 2010). Similar behavior, i.e., preferential and stronger binding of a chosen phosphoinositide in the presence of other phospholipids, mostly in the form of micelles was also described for proteins bearing pleckstrin homology domain (Sugiki et al., 2012). An important role of different phospholipids for the interaction of HIV-1 MA with the PM has been proposed recently by Vlach and Saad (2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane (PM) through its N-terminal domain, the matrix protein (MA). However, single genera of Retroviridae family differ in the way how they interact with PM. While in case of Lentiviruses (e.g., human immunodeficiency virus) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, Betaretroviruses [Mason-Pfizer monkey virus (M-PMV)] first assemble their virus-like particles (VLPs) in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV MA with PM with emphasis on the structural aspects of the interaction with single phospholipids.
    Frontiers in Microbiology 01/2013; 4:423. DOI:10.3389/fmicb.2013.00423 · 3.99 Impact Factor
Show more


9 Reads
Available from