Photon Factory Activity Report 2018 #36 (2019)

BL-1A, BL-17A/ 2018RP-12

Structural study on Atg2 that mediates autophagosomal membrane expansion
1Takuo OSAWA1 and Nobuo N. NODA1,*
Institute of Microbial Chemistry, Microbial Chemistry Research Foundation,
3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

1 Introduction
Autophagy is an intracellular degradation systemconserved among eukaryotes. When autophagy is induced,
a membrane structure called an isolation membrane (IM)suddenly appears in the cytoplasm, which expands andseals into a double membrane organelle, anautophagosome, where a portion of the cytoplasm issequestered. Autophagosome then fuses with the lysosome
or vacuole and the inner contents are degraded bylysosomal hydrolases [1]. For autophagosome formation,
IMs must receive phospholipids from membrane-source
organelles, such as ER, as building blocks. However, themechanism of how phospholipids are transported to the IM
through aqueous cytoplasm remained to be elucidated.
Atg2 is one of the core Atg proteins that mediate
autophagosome formation and localizes to the expandingedge of the IM [2]. Since Atg2 also contacts with the ER[2], it was proposed that Atg2 functions as a tether
between the ER and the IM. However, the molecularfunctions of Atg2 have been unknown due to its low
sequence homology with characterized proteins. Overall
structure of human Atg2 was reported by negativestaining electron microscopy at low resolution, whichshowed a club-like architecture [3, 4]. Here, in order toobtain high-resolution structural data on Atg2, we
crystallized the N-terminal region (NR) of Atg2 anddetermined the crystal structure [5].
2 ExperimentS. pombe Atg2NR was expressed in E. coli as a fusion
protein with a T4 lysozyme and a His-tag. After affinity
purification using a TALON resin column, the protein wassubjected to size-exclusion chromatography using aSuperdex 200 column. For preparation of PE-bound form,
fusion protein was mixed with PE in a 1:2 molar ratio for
1 h before size-exclusion chromatography. Purified fusion
protein was crystallized by the hanging drop vapour
diffusion method using 15-16% PEG400, 0.1 M Bis-Tris(pH 6.5), and 0.2 M ammonium sulfate as a reservoirsolution. The crystals were soaked in the reservoir solution
containing 30% PEG400 and 2% trehalose as acryoprotectant. X-ray diffraction data were collected at
BL-1A and BL-17A of the Photon Factory, and processedusing HKL2000. Initial phasing was performed by a
combination of the molecular replacement and single
wavelength anomalous dispersion methods using AutoMRand AutoSol in Phenix. Models were built manually with
COOT and crystallographic refinement was performedwith Phenix.
3 Results and Discussion

Data collection and refinement statistics aresummarized in Table 1. Atg2NR is comprised of a twisted
-sheet and a helical region, which together assume aglobular structure with a large hydrophobic cavity (Fig. 1).
The structure of the Atg2NR-PE complex reveals that thehydrophobic cavity accommodates the acyl portions of thePE. Since this interaction mode is similar to other lipid
transfer proteins, we studied the lipid transfer activity ofAtg2 in vitro and revealed that Atg2 actually possesses
lipid transfer activity between liposomes. Moreover, frommutational studies of Atg2 both in vitro and in vivo, weproposed a model that Atg2 tethers the ER to the IM and at
the same time mediates phospholipid transfer from the ER
to the IM for expanding the IM into the autophagosome [5,6].
Table 1: Data collection and refinement statistics
T4L-SpAtg2NR
PE-bound form
(PDB 6A9E)(PDB 6A9J)
Data collection
Space group
P21
P21
Cell dimensions
a, b, c (Å)
86.5, 62.7, 86.3
86.7, 62.9, 86.7
90.0, 91.8, 90.0
90.0, 91.9, 90.0   ()Peak
Wavelength
0.9692
Resolution (Å)a50-3.20
50-2.70(3.26-3.20)a(2.75-2.70)
Rmerge0.145 (0.579)
0.381 (0.439)
16.8 (3.4)
23.8 (2.6)
I/(I)
Completeness (%) 100 (100)99.9 (99.4)
Redundancy
13.7 (14.3)
6.6 (6.2)
Refinement
Resolution (Å)
44-3.238-2.7
No. reflections
29590
25884
Rwork / Rfree
0.224/0.266
0.251/0.268
No. atoms
Protein
5023
4998
PE
102
B factors
Protein
63.0
67.8
PE
68.2
R.m.s deviations
Bond lengths (Å) 0.003
0.003
0.863
Bond angles () 0.644
aValues in parentheses are for highest-resolution shell.

Photon Factory Activity Report 2018 #36 (2019)

Fig. 1: Structure of Atg2NR complexed with PE.
Acknowledgement
We thank Y. Ishii for her help with protein expression. S.
pombe Atg2 cDNA was provided by the RIKEN BRC
through the National BioResource Project of theMEXT/AMED, Japan. This work was supported by Japan
Science and Technology Agency (JST) CREST and theJapan Society for the Promotion of Science (JSPS)
KAKENHI.
References[1] Noda, N. N. and Inagaki, F. Annu Rev Biophys 44, 101(2015).
[2] Suzuki, K. et al. J. Cell Sci. 126, 2534 (2013).
[3] Chowdhury, S. et al. Proc. Natl. Acad. Sci. U. S. A. 115,
E9792 (2018).
[4] Zheng, J. X. et al. Autophagy 13, 1870 (2017).
[5] Osawa, T. et al. Nat. Struct. Mol. Biol. 26, 281 (2019).
[6] Osawa, T. and Noda, N. N. Protein Sci. 28, 1005 (2019).
* nn@bikaken.or.jp