hfAIM: A reliable bioinformatics approach for in silico genome-wide identification of autophagy-associated Atg8-interacting motifs in various organisms

Abstract

Most of the proteins that are specifically turned over by selective autophagy are recognized by the presence of short Atg8 interacting motifs (AIMs) that facilitate their association with the autophagy apparatus. Such AIMs can be identified by bioinformatics methods based on their defined degenerate consensus F/W/Y-X-X-L/I/V sequences in which X represents any amino acid. Achieving reliability and/or fidelity of the prediction of such AIMs on a genome-wide scale represents a major challenge. Here, we present a bioinformatics approach, high fidelity AIM (hfAIM), which uses additional sequence requirements-the presence of acidic amino acids and the absence of positively charged amino acids in certain positions-to reliably identify AIMs in proteins. We demonstrate that the use of the hfAIM method allows for in silico high fidelity prediction of AIMs in AIM-containing proteins (ACPs) on a genome-wide scale in various organisms. Furthermore, by using hfAIM to identify putative AIMs in the Arabidopsis proteome, we illustrate a potential contribution of selective autophagy to various biological processes. More specifically, we identified 9 peroxisomal PEX proteins that contain hfAIM motifs, among which AtPEX1, AtPEX6 and AtPEX10 possess evolutionary-conserved AIMs. Bimolecular fluorescence complementation (BiFC) results verified that AtPEX6 and AtPEX10 indeed interact with Atg8 in planta. In addition, we show that mutations occurring within or nearby hfAIMs in PEX1, PEX6 and PEX10 caused defects in the growth and development of various organisms. Taken together, the above results suggest that the hfAIM tool can be used to effectively perform genome-wide in silico screens of proteins that are potentially regulated by selective autophagy. The hfAIM system is a web tool that can be accessed at link:

Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics

“…The residual matrix protein import observed in pex14 null mutants [46] implies important roles for the PEX13 docking peroxin, which might be explored using viable pex13 partial loss-of-function alleles [53,86]. Moreover, some peroxins, such as PEX10, are implicated in processes in addition to matrix protein import [76,77,80] that may be plant-specific. We are only beginning to understand the roles of peroxules in peroxisome metabolism and inter-organellar communication.…”

“…Intriguingly, Arabidopsis PEX10 and PEX6 interact with AUTOPHAGY-RELATED8 (ATG8) [76], the ubiquitin-like protein that decorates the nascent autophagosome membrane to mediate autophagic cargo engulfment [reviewed in 66]. This interaction suggests that certain peroxins might promote pexophagy in addition to their roles in matrix protein import.…”

“…This interaction suggests that certain peroxins might promote pexophagy in addition to their roles in matrix protein import. Strikingly, a single amino acid change (G93E) in PEX10 adjacent to the PEX10-ATG8 interaction domain [76] confers seedling lethality when homozygous and aberrant peroxisome morphology when heterozygous [77]. PEX10 carrying this G93E mutation still interacts with ATG8 in bimolecular fluorescence complementation experiments; the additional negative charge suggests that PEX10-ATG8 binding would be increased, perhaps causing excessive targeting of peroxisomes to the autophagy machinery [76].…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…Intriguingly, the Arabidopsis RING peroxin PEX10 and the ATPase PEX6 bind ATG8 in bimolecular fluorescence complementation assays (Xie et al, 2016). Moreover, Arabidopsis DSK2, a ubiquitin-binding protein that interacts with the RING domains of PEX2 and PEX12 , also binds ATG8 and promotes selective autophagy of a growth-promoting transcription factor (Nolan et al, 2017).…”

“…Moreover, mammalian PEX3 and PEX13 promote autophagy of mitochondria (mitophagy) whereas PEX19 and PEX14 are necessary for general autophagy (Lee et al, 2017). The dual roles of peroxins acting in biogenesis and to attract autophagy machinery (Zutphen et al, 2008;Motley et al, 2012;Xie et al, 2016) hint at mechanisms to trigger peroxisome degradation when import becomes dysfunctional. These discoveries highlight the intimate relationships among organelles and prompt the question of whether plant peroxins are similarly promiscuous.…”