Molecular determinants regulating selective binding of autophagy adapters and receptors to ATG8 proteins.

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.

Measurement of the Activity of the Atg4 Cysteine Proteases.

While only one Atg4 is present in yeast, there are four Atg4 homologues in human and in mouse with different substrate specificities and catalytic efficiencies. The molecule Atg4 is a type of cysteine protease, and is known for its crucial roles in cleavage of the Atg8 family proteins before they can be conjugated to phospholipids, and also in cleavage of the conjugated Atg8 molecules from the membrane, a process known as deconjugation. Both processes are required for the maximal efficiency in autophagosome biogenesis. Atg4 could thus be a target for intervention of the autophagy process. It is thus important to measure Atg4 activity to determine and to modulate the autophagy function. Here, we review the catalytic functions and regulatory mechanisms of human Atg4 proteases and discuss the methodology for analyzing Atg4 activity in details.

Biochemical Analysis of Autophagy in Algae and Plants by Monitoring the Electrophoretic Mobility of ATG8.

Identification of specific autophagy markers has been fundamental to investigate autophagy as catabolic process. Among them, the ATG8 protein turned out to be one of the most widely used and specific molecular markers of autophagy both in higher and lower eukaryotes. Here, we describe how ATG8 can be used to monitor autophagy in Chlamydomonas and Arabidopsis by western blot analysis.

Detection of Autophagy in Plants by Fluorescence Microscopy.

Autophagy is a key process for degradation and recycling of proteins or organelles in eukaryotes. Autophagy in plants has been shown to function in stress responses, pathogen immunity, and senescence, while a basal level of autophagy plays a housekeeping role in cells. Upon activation of autophagy, vesicles termed autophagosomes are formed to deliver proteins or organelles to the vacuole for degradation. The number of autophagosomes can thus be used to indicate the level of autophagy. Here, we describe two common methods used for detection of autophagosomes, staining of autophagosomes with the fluorescent dye monodansylcadaverine, and expression of a fusion between GFP and the autophagosomal membrane protein ATG8.