Atg11-mediated activation of Atg1 kinase in fission yeast

ABSTRACT

The protein kinase Atg1 is a key player in macroautophagy/autophagy, but how its activity is regulated in various organisms is inadequately understood. Our recent study showed that in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity depends on Atg11, but not Atg13, Atg17, or Atg101. Notably, a 62 amino acid region of S. pombe Atg11 is sufficient for activating Atg1. This region is composed of two parts: an Atg1-binding domain and a homodimerization domain. Atg11 uses this region to dimerize Atg1. Dimerized Atg1 is activated through cis-autophosphorylation.

Macroautophagy (hereafter autophagy) maintains cellular homeostasis by transporting cytoplasmic materials into lysosomes and vacuoles for recycling. Autophagy is driven by a set of core autophagy-related (Atg) proteins, which are largely conserved across eukaryotes. Among the core Atg proteins, the only one with a protein kinase activity is Atg1 (called ULK1 in mammals). Atg1 kinase activity is essential for autophagy.

In the budding yeast Saccharomyces cerevisiae where autophagy has been best studied, Atg1 kinase activity is controlled by several other core Atg proteins. In starvation-induced nonselective bulk autophagy, Atg13 and Atg17, two scaffold proteins, act together to activate Atg1. In the cytoplasm-to-vacuole targeting (Cvt) pathway, which is a selective autophagy pathway that operates under nutrient-rich conditions, Atg13 and another scaffold protein, Atg11, are required for activating Atg1 kinase. The role of scaffold proteins in activating Atg1 is thought to be in promoting the oligomerization of Atg1 so that this protein can undergo concentration-dependent autophosphorylation. It has been assumed that Atg1 autophosphorylation occurs via trans-autophosphorylation. However, no experimental evidence supporting this assumption exists.

Atg11 is ubiquitously present in eukaryotic organisms, and its mammalian ortholog is called RB1CC1/FIP200. Even though Atg11 is dispensable for nonselective bulk autophagy in S. cerevisiae, in many other organisms, including the fission yeast Schizosaccharomyces pombe, Drosophila, Arabidopsis, and mammals, Atg11 is crucial for bulk autophagy. The exact essential roles of Atg11 in bulk autophagy in these organisms have been unclear.

In a recently published study, we showed that in S. pombe, the essential role of Atg11 in bulk autophagy is to activate the kinase activity of Atg1 [1]. We analyzed the kinase activity of S. pombe Atg1 using an in vitro kinase assay, in which both Atg1 autophosphorylation and the phosphorylation of an artificial consensus site substrate were used as readouts. Unexpectedly, we found that Atg1 kinase activity did not obviously change when Atg13, Atg17, or an Atg13-interacting protein, Atg101, were absent, whereas Atg1 kinase activity was largely abolished when Atg11 was absent.

To understand how S. pombe Atg11 promotes Atg1 kinase activity, we performed truncation analysis on Atg11 and found that, remarkably, a 62 amino acid fragment, Atg11[522–583], which does not overlap with any of three database-annotated domains in Atg11 (a ubiquitin-like domain, an Atg17-like domain, and an Atg11 domain), is the minimal region sufficient for supporting Atg1 kinase activity and for rescuing the autophagy defect of the atg11Δ mutant. We further showed that this fragment can be functionally divided into two parts. The first part, Atg11[522–544], is responsible for interacting with Atg1, and two conserved aromatic residues within it, F526 and Y527, are essential for Atg1 binding and for the autophagy function of Atg11. The second part, Atg11[546–583], mediates a self-interaction of Atg11. Gel filtration analysis showed that Atg11[522–583] forms a homodimer. The autophagy function of Atg11 can be bypassed by adding a homodimerization motif to Atg1. Together, these findings demonstrated that the main role of S. pombe Atg11 in bulk autophagy is to dimerize Atg1 and thereby activate its kinase activity.

S. cerevisiae Atg11 and human RB1CC1 interact with selective autophagy receptors using their C-terminal regions containing the conserved Atg11 domain. Interestingly, in S. pombe, Atg11[522–583], despite lacking the Atg11 domain, is capable of supporting DTT-induced reticulophagy, the only known selective autophagy pathway in fission yeast. Thus, the role of Atg11 in connecting selective autophagy receptors to other core Atg proteins may not be always conserved.

To understand how dimerization-induced Atg1 activation occurs, we used the heterodimerizing protein pair GFP and GFP-binding protein (GBP) to artificially dimerize Atg1 and bypass Atg11. This setup allowed us to determine whether both Atg1 molecules in a dimer need to be catalytically active, as predicted by the trans-autophosphorylation model. Contrary to the prediction, we found that autophagy was unaffected when one of the two Atg1 molecules in a dimer was catalytically inactive, indicating that Atg1 activation can be achieved through cis-autophosphorylation. This finding sheds new light on the mechanistic details of Atg1 activation.

Disclosure statement

The authors declare no conflict of interest.