Autophagy is a potent cellular degradation pathway, and its activation needs to be tightly controlled. Cargo receptors mediate selectivity during autophagy by bringing cargo to the scaffold protein ...Atg11 and, in turn, to the autophagic machinery, including the central autophagy kinase Atg1. Here we show how selective autophagy is tightly regulated in space and time to prevent aberrant Atg1 kinase activation and autophagy induction. We established an induced bypass approach (iPass) that combines genetic deletion with chemically induced dimerization to evaluate the roles of Atg13 and cargo receptors in Atg1 kinase activation and selective autophagy progression. We show that Atg1 activation does not require cargo receptors, cargo-bound Atg11, or Atg13 per se. Rather, these proteins function in two independent pathways that converge to activate Atg1 at the vacuole. This pathway architecture underlies the spatiotemporal control of Atg1 kinase activity, thereby preventing inappropriate autophagosome formation.
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•Atg1 activation in selective autophagy is tightly regulated in space and time•Atg1 is activated by the convergence of two independent pathways at the vacuole•Cargo receptors and Atg13 function as tethers for Atg11 and Atg1, respectively•Both cargo receptors and Atg13 can be functionally bypassed in selective autophagy
Applying a newly developed synthetic bypass approach (iPass), Torggler et al. show that in selective autophagy, Atg1 kinase is activated at the vacuole by the convergence of two independent pathways. This allows the spatiotemporal control of Atg1 kinase activation.
Autophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating ...autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.
N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in
Escherichia coli
, in particular in proteins bearing an N-terminal histidine-tag. This ...post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of
13
C/
15
N labeled proteins, their chemical shifts were not yet reported. Here we present the complete
1
H and
13
C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His)
6
. In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.