Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

Loss of Elongator- and KEOPS-Dependent tRNA Modifications Leads to Severe Growth Phenotypes and Protein Aggregation in Yeast.

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.

Vac8 spatially confines autophagosome formation at the vacuole in S. cerevisiae.

Autophagy is initiated by the formation of a phagophore assembly site (PAS), the precursor of autophagosomes. In mammals, autophagosome formation sites form throughout the cytosol in specialized subdomains of the endoplasmic reticulum (ER). In yeast, the PAS is also generated close to the ER, but always in the vicinity of the vacuole. How the PAS is anchored to the vacuole and the functional significance of this localization are unknown. Here, we investigated the role of the PAS-vacuole connection for bulk autophagy in the yeast Saccharomyces cerevisiae We show that Vac8 constitutes a vacuolar tether that stably anchors the PAS to the vacuole throughout autophagosome biogenesis via the PAS component Atg13. S. cerevisiae lacking Vac8 show inefficient autophagosome-vacuole fusion, and form fewer and smaller autophagosomes that often localize away from the vacuole. Thus, the stable PAS-vacuole connection established by Vac8 creates a confined space for autophagosome biogenesis between the ER and the vacuole, and allows spatial coordination of autophagosome formation and autophagosome-vacuole fusion. These findings reveal that the spatial regulation of autophagosome formation at the vacuole is required for efficient bulk autophagy.

Absolute Quantification of Noncoding RNA by Microscale Thermophoresis.

Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization-based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA-Seq-based quantification approaches, strongly suggesting a bias due to tRNA modifications in the latter. Further applications include the quantification of rRNA as well as of polyA levels in cellular RNA.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.