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Methods Mol Biol. 2018;1694:215-223. doi: 10.1007/978-1-4939-7398-9_20.

Analysis of the Intracellular Localization of Transiently Expressed and Fluorescently Labeled Copper-Containing Amine Oxidases, Diamine Oxidase and N-Methylputrescine Oxidase in Tobacco, Using an Agrobacterium Infiltration Protocol.

Author information

1
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, 8916-5, Ikoma, Japan. t-shouji@bs.naist.jp.

Abstract

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.

KEYWORDS:

Copper-containing amine oxidase; Diamine oxidase; Infiltration; N-methylputrescine oxidase; Nicotiana benthamiana; Nicotine biosynthesis; Peroxisome; Polyamine catabolism; Tobacco; Transient expression; Yellow fluorescent protein; mCherry

PMID:
29080170
DOI:
10.1007/978-1-4939-7398-9_20
[Indexed for MEDLINE]

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