We previously developed a web server CPGAVAS for annotation, visualization and GenBank submission of plastome sequences. Here, we upgrade the server into CPGAVAS2 to address the following challenges: (i) inaccurate annotation in the reference sequence likely causing the propagation of errors; (ii) difficulty in the annotation of small exons of genes petB, petD and rps16 and trans-splicing gene rps12; (iii) lack of annotation for other genome features and their visualization, such as repeat elements; and (iv) lack of modules for diversity analysis of plastomes. In particular, CPGAVAS2 provides two reference datasets for plastome annotation. The first dataset contains 43 plastomes whose annotation have been validated or corrected by RNA-seq data. The second one contains 2544 plastomes curated with sequence alignment. Two new algorithms are also implemented to correctly annotate small exons and trans-splicing genes. Tandem and dispersed repeats are identified, whose results are displayed on a circular map together with the annotated genes. DNA-seq and RNA-seq data can be uploaded for identification of single-nucleotide polymorphism sites and RNA-editing sites. The results of two case studies show that CPGAVAS2 annotates better than several other servers. CPGAVAS2 will likely become an indispensible tool for plastome research and can be accessed from http://www.herbalgenomics.org/cpgavas2.

Summary Autophagy is a process of cellular self-digestion induced by various forms of starvation. The mechanisms by which metabolic deficiencies are sensed by a cell to regulate autophagy remain unclear. While nitrogen deficit is a common trigger, yeast cells induce autophagy upon switch from a rich to minimal media without nitrogen starvation. We show that the amino acid methionine is sufficient to inhibit such non-nitrogen starvation (NNS)-induced autophagy. Methionine boosts synthesis of the methyl donor, S-adenosylmethionine (SAM). SAM inhibits autophagy and promotes growth through the action of the methyltransferase Ppm1p, which methylates the catalytic subunit of PP2A in tune with SAM levels. Methylated PP2A promotes dephosphorylation of Npr2p, a component of a conserved complex that regulates NNS-autophagy and other growth-related processes. Thus, methionine and SAM levels represent a critical gauge of amino acid availability that is sensed via this distinctive methylation modification of PP2A to reciprocally regulate cell growth and autophagy.

SUMMARY Protein translation is an energetically demanding process that must be regulated in response to changes in nutrient availability. Herein, we report that the thiolation status of wobble-uridine (U34) nucleotides present on lysine, glutamine or glutamate tRNAs reflects intracellular methionine and cysteine availability, and regulates cellular translational capacity and metabolic homeostasis. tRNA thiolation is important for growth under nutritionally challenging environments and required for efficient translation of genes enriched in lysine, glutamine, and glutamate codons, which frequently encode proteins important for translation and growth-specific processes. tRNA thiolation is down-regulated during sulfur starvation in order to decrease sulfur consumption and growth, and its absence leads to a compensatory increase in enzymes involved in methionine, cysteine, and lysine biosynthesis. Thus, tRNA thiolation enables cells to modulate translational capacity according to the availability of sulfur amino acids, establishing a functional significance for this conserved tRNA nucleotide modification in cell growth control.

Autophagy can be induced in response to more subtle changes in nutritional state, without severe nitrogen starvation. The Iml1p-Npr2p-Npr3p complex selectively regulates this form of autophagy.

Mitochondria-associated degradation (MAD) mediated by the Cdc48 complex and proteasome degrades ubiquitinated mitochondrial outer-membrane proteins. MAD is critical for mitochondrial proteostasis, but it remains poorly characterized. We identified several mitochondrial Cdc48 substrates and developed a genetic screen assay to uncover regulators of the Cdc48-dependent MAD pathway. Surprisingly, we identified Doa1, a substrate-processing factor of Cdc48 that inhibits the degradation of some Cdc48 substrates, as a critical mediator of the turnover of mitochondrial Cdc48 substrates. Deletion of DOA1 causes the accumulation and mislocalization of substrates on mitochondria. Profiling of Cdc48 cofactors shows that Doa1 and Cdc48-Ufd1-Npl4 form a functional complex mediating MAD. Biochemically, Doa1 interacts with ubiquitinated substrates and facilitates substrate recruitment to the Cdc48-Ufd1-Npl4 complex. Functionally, Doa1 is critical for cell survival under mitochondrial oxidative stress, but not ER stress, conditions. Collectively, our results demonstrate the essential role of the Doa1–Cdc48-Ufd1-Npl4 complex in mitochondrial proteostasis and suggest that Doa1 plays dual roles on the Cdc48 complex.