title: Expression of Malus xiaojinensis IRT1 (MxIRT1) protein in transgenic yeast cells leads to degradation through autophagy in the presence of excessive iron.
abstract: Iron is essential for plants, but highly toxic when present in excess. Consequently, iron uptake by root transporters must be finely tuned to avoid excess uptake from soil under iron excess. The iron-regulated transporter of Malus xiaojinensis (MxIRT1), induced in roots under iron deficiency, is a highly effective iron(II) transporter. Here, we investigated how the presence of excessive iron leads to MxIRT1 degradation in yeast expressing this plant iron transporter protein. To determine the relationship between iron abundance and MxIRT1 degradation, relative levels of autophagy-related gene-8 (ATG8) mRNA and the active ATG8-phosphatidylethanolamine-conjugated (PE) protein were measured in wild-type yeast and the autophagic mutant strains atg1∆, atg5∆, atg7∆, ypt7∆ and tor1∆ under normal and excessive iron conditions. The data showed that the exposure of MxIRT1-eGFP-transformed wild-type and tor1∆ strains to excessive iron led to significantly increased levels of ATG8 transcript and ATG8-PE protein, which resulted in enhanced MxIRT1 degradation. Co-localization of mCherry-ATG8 and MxIRT1-eGFP provided evidence that these proteins interact during autophagy in yeast. While inhibition of autophagic initiation, autophagosome formation and vacuole fusion all decreased MxIRT1 degradation. PMSF inhibition of autophagy prevented degradation, leading to the accumulation of MxIRT1-containing vesicles in the vacuoles. MxIRT1-vesicles were sorted into autophagosomes for iron-induced degradation in yeast, whereas the endogenous iron(II) transporter Fet4 was degraded in an autophagy-independent manner. Moreover, immunoprecipitation showed that multimono-ubiquitins provided MxIRT1 with the ubiquitination signal. Together, three factors, iron excess, autophagy and mono-ubiquitination, affect the functional activity and stability of exogenous MxIRT1 in yeast, thereby preventing iron uptake via this root transporter.
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Shuang
authors: S
authors: Li
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Xi
authors: X
authors: Zhang
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Xiu-Yue
authors: XY
authors: Zhang
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.|Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
authors: Wei
authors: W
authors: Xiao
authors: Department of Biological Sciences, State University of New York, Buffalo, NY, USA.
authors: James O
authors: JO
authors: Berry
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Peng
authors: P
authors: Li
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Si
authors: S
authors: Jin
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Song
authors: S
authors: Tan
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Peng
authors: P
authors: Zhang
authors: Institute of Mathematics and Interdisciplinary Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Wei-Zhong
authors: WZ
authors: Zhao
authors: College of Life Sciences, Capital Normal University, Beijing, People's Republic of China.
authors: Li-Ping
authors: LP
authors: Yin
mesh_terms: D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020869:Gene Expression Profiling; D007501:Iron / Q000378:metabolism*; D027845:Malus / Q000235:genetics*; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D059748:Proteolysis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012440:Saccharomyces / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics; D054875:Ubiquitination
chemical_list: C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D027682:Cation Transport Proteins; D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D007501:Iron
keywords: Fet4; MxIRT1; autophagy; iron excess; mono-ubiquitination; transgenic yeast