Biochemical and Biophysical Research Communications
Structural and functional insight into the N-terminal domain of the clathrin adaptor Ent5 from Saccharomyces cerevisiae
Introduction
Clathrin dependent protein trafficking is ubiquitous at many cellular compartments of all eukaryotic cells. The clathrin coated vesicles (CCVs) mediate either endocytic traffic originated at plasma membrane (PM) or biosynthetic traffic originated at trans-Golgi network (TGN) [1], [2]. Intensive studies in both mammalian and yeast cells have uncovered more than 40 proteins involved in clathrin-mediated vesicles trafficking between the TGN and endosomes [3], indicating the importance and complexity of the vesicular trafficking system. It is known that clathrin adaptors such as adaptor proteins (APs) and Golgi-localized, γ-ear containing, ADP-ribosylation factor-binding proteins (GGAs) play essential roles in coat assembly, clathrin binding and cargo-sorting [4]. However, detailed processes of CCV formation remain unclear.
In Saccharomyces cerevisiae, there are 5 clathrin adaptors at TGN, including ScAP-1 complex, ScGGA1/ScGGA2, two ENTH domain-containing proteins ScEnt3 and ScEnt5. The phosphoinositide-binding proteins ScEnt3 and ScEnt5 are required for protein transport from the TGN to the endosome [5], and both proteins associate with γ-ear domains of clathrin adaptor protein ScGGA2 and ScAP-1 in vivo [6]. Deletion of individual ENT3 or ENT5 does not display trafficking defects in clathrin-mediated transport but cells lacking both ENT3 and ENT5 affect the sorting of proteins between the TGN and the endosomes (including CPY, CPS and α-factor maturation) as well as the trafficking of multivesicular bodies (MVB) [5], [7]. These results suggest that ScEnt3 and ScEnt5 serve as partially redundant adaptors in clathrin-mediated TGN-endosome traffic. However, ScEnt3 and ScEnt5 proteins also display some specific functions. On clathrin-binding properties, ScEnt5 has two canonical clathrin-binding motifs and can be co-immunoprecipitated with clathrin, whereas ScEnt3 lacks. ScEnt3 acts principally in the GGA-mediated TGN sorting and only the ENTH domain of ScEnt3 binds to the endosomal SNAREs Vti1, Pep12 and Syn8 [8]. ScEnt5 acts principally in both the GGA and AP-1 mediated TGN sorting, and it is vital for the AP-1-mediated pathway [7]. ScEnt5 (but not ScEnt3) binds to the multi-pass transmembrane protein chitin synthase ScChs3, but ScEnt3 and ScEnt5 are both required for its trafficking from the Golgi [5].
The ScEnt3 N-terminal domain contains an EpsinR N-terminal homolog (ENTH) signature motif. In contrast to ScEnt3, ScEnt5 carries a lysine-rich AP180 N-terminal homolog (ANTH)-like phosphoinositide-binding motif. Thus, ScEnt5 is unlikely to promote membrane curvature and might function more like hAP180 than hEpsin1 [9]. However, because lack of the sufficient structural and functional evidence, both of the N-terminal domain classification and the phosphoinositide binding specificity of ScEnt5 are still vague. In the present work, three crystal structures of the Ent5 N-terminal domain from the Saccharomyces cerevisiae were reported at resolutions of 2.10, 1.80 and 2.20 Å respectively. The structure contains an insertion α′ helix that does not exist in other ENTH or ANTH domains. Furthermore, we investigated the classification of ScEnt5-N31−191 by evolutionary history analyses and structure comparisons, and found the ScEnt5 N terminal domain showed different phosphoinositide binding property with rEpsin1 and rCALM. Our studies facilitate the understanding of the molecular mechanisms of ScEnt5 associated clathrin-mediated vesicles trafficking.
Section snippets
Cloning, overexpression and purification
The coding DNA sequence for full length Ent5 (NM_001180460.3) was obtained from genome DNA of Saccharomyces cerevisiae S288c by using Prime STAR HS DNA Polymerase (TaKaRa); The Shorter DNA fragments were amplified from full length DNA. The DNA fragment was cloned into a modified pET-22b(+) vector with a C-terminal 6×His tag by using the BamHI/XhoI restriction site. Recombinant protein was expressed in E. coli Rosetta2 (DE3) (Novagen). The bacterial cultures were harvested by centrifugation
Overall structure of ScEnt5 N-terminal domain
We constructed plasmids for full-length Ent5 from S. cerevisiae and for the N-terminal domain of ScEnt5 (Glu31-Ser213) (ScEnt5-N31−213). However, owing to the poor solubility and stability problems of full-length ScEnt5, only ScEnt5-N31−213 could be purified for crystallization. After half a year, ScEnt5-N31−213 could be crystallized in one crystallization condition contaminated by bacteria. A diffraction data set was collected to 2.10 Å resolution for ScEnt5-N31−213 (designated as ScEnt5-Native
Acknowledgements
The authors thank the staff of BL17U at SSRF for assistance with synchrotron diffraction data collection. Financial support for this project was provided by the Chinese National Natural Science Foundation (Grant No. 31130018), the Chinese Ministry of Science and Technology (Grant No. 2012CB917200), the Chinese National Natural Science Foundation (Grant Nos. 31370732, 31270014 and U1432107), the Scientific Research Grant of Hefei Science Center of CAS (Grant No. 2015SRG-HSC042).
References (28)
- et al.
The mechanisms of vesicle budding and fusion
Cell
(2004) - et al.
From endosomes to the trans-Golgi network
Semin. Cell Dev. Biol.
(2014) - et al.
ENTH/ANTH domains expand to the Golgi
Trends Cell Biol.
(2003) - et al.
Processing of X-ray diffraction data collected in oscillation mode
Method Enzymol.
(1997) - et al.
PRIP, a novel Ins(1,4,5)P3 binding protein, functional significance in Ca2+ signaling and extension to neuroscience and beyond
Mol. Cells
(2005) - et al.
Biological basket weaving: formation and function of clathrin-coated vesicles
Annu. Rev. Cell Dev. Biol.
(2001) - et al.
Adaptors for clathrin coats: structure and function
Annu. Rev. Cell Dev. Biol.
(2004) - et al.
Ent3p and Ent5p exhibit cargo-specific functions in trafficking proteins between the trans-Golgi network and the endosomes in yeast
Mol. Biol. Cell
(2007) - et al.
Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif
Nat. Cell Biol.
(2003) - et al.
Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p
Mol. Biol. Cell
(2006)