Abstract

Article info 2017 5 (3)    005  (03)   pp.  114 ~ 117
Title Purification, crystallization, and X-ray crystallographic analysis of Vac8p complexed with Atg13p from Saccharomyces cerevisiae
Authors Jumi Park1,4, Kyonghwa Song2, Sohyeon Oh2, Taewon Son2, Jun Lee2, Ayoung Park2, Hyun Ji Kim2, Youngsoo Jun3,4 and Changwook Lee1,4*
Institutions 1Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, 50 UNIST-gil, Ulsan 44919, Republic of Korea, 2Busan Science High School, 455-1 Geumsaem-ro, Busan 46235, Republic of Korea, 3School of Life Sciences and 4Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea. *Correspondence: changwook@unist.ac.kr
Abstract Vac8p is a vacuolar protein that plays pivotal roles in both vacuole inheritance and the formation of nucleus vacuole junction (NVJ) in yeast. The Vac8p directly interacts with Atg13p, a component of the autophagy machinery, and mediates cytoplasm-to-vacuole targeting (Cvt) pathway, resulting in the maturation of aminopeptidase I (Ape1p). Here, we coexpressed and purified Saccharomyces cerevisiae Vac8p complexed with Atg13p in Escherichia coli bacteria cells, and crystallized the complex proteins under the condition of 25% (v/v) PEG 400, 100 mM Tris pH 8.5, 2% (v/v) Ethylene glycol, 2% (w/v) PEG 3350, 1.5% (w/v) PEG 20000, 5 mM DTT at 293K. X-ray diffraction data of the crystals were collected to 2.9 Å resolution at the synchrotron radiation. The crystals belong to the orthorhombic space group P212121 with unit cell parameters a = 62.7 Å, b = 92.4 Å, and c = 139.9 Å. The asymmetric unit contains one Vac8p-Atg13p heterodimer with a corresponding VM of 2.92 Å3 Da-1 and solvent content of 57.8%.