Caiji Gao, Xiaohong Zhuang, Yong Cui, Xi Fu, Yilin He, Qiong Zhao, Yonglun Zeng, Jinbo Shen, Ming Luo, and Liwen Jiang

Significance

Macroautophagy (hereafter as autophagy) involves the delivery of cytosolic materials via autophagosome upon its fusion with the endosome and lysosome/vacuole. The endosomal sorting complex required for transport (ESCRT) machinery is responsible for the formation of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) and the sorting of ubiquitinated membrane cargos into MVB ILVs for degradation. Here, we show that, in addition to regulating MVB biogenesis, the plant-specific ESCRT component FYVE domain protein required for endosomal sorting 1 (FREE1) also plays dual roles in vacuolar protein transport and autophagic degradation. FREE1 directly interacts with a plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 to manipulate the autophagic degradation in plants. Thus, we demonstrate multiple functions of FREE1 and a direct link between the ESCRT machinery and autophagy process in plants.

Abstract

Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

See: http://www.pnas.org/content/112/6/1886.abstract.html?etoc

PNAS February 10, 2015; vol.112; no. 6 1886-1891  

Fig. 1. FREE1 regulates the transport of vacuolar proteins to protein storage vacuoles. (A) The free1 mutant is defective in the transport of seed storage proteins. GFP-CT24 was expressed in the wild-type (WT) and free1 (^+/−) heterozygous plants. The seeds of the F2 population were inspected with a fluorescence microscope. free1/GFP-CT24 produced green fluorescent seeds (arrows). (B) The GFP-CT24 is targeted to protein storage vacuoles in wild-type seed cells, whereas it is secreted to the extracellular spaces in free1 mutant cells. (Scale bars, 10 μm.) (C) The free1 mutant is defective in processing of seed storage proteins. The wild-type seeds and the selected green fluorescent seeds shown in A were subjected to protein extraction for Coomassie blue staining or immunoblot analysis with GFP and 12S globulin antibodies. Note the accumulation of precursors of storage proteins in free1 mutant seeds.