Purification, crystallization, and X-ray crystallographic analysis of Vac8p complexed with Atg13p from Saccharomyces cerevisiae

• 발행기관: 한국구조생물학회
• 저자명: Jumi Park Kyonghwa Song Sohyeon Oh Taewon Son Jun Lee Ayoung Park Hyun Ji Kim Youngsoo Jun Changwook Lee
• 간행물 정보: 『Biodesign』Vol 5, No 3, Sep, 114~117쪽, 전체 4쪽
• 주제분류: 자연과학 > 생물학
• 파일형태: PDF
• 발행일자: 2017.09.30

영문 초록

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 P2 1 2 1 2 1 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 V M of 2.92 Å 3 Da -1 and solvent content of 57.8%.

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INTRODUCTION

RESULTS AND DISCUSSION