Metamorphosis by ATG13 and ATG101 in human autophagy initiation.

ABBREVIATIONS: ATG, Autophagy-related, HORMA, protein domain named after HOP1-MAD2-REV7; RB1CC1, RB1 inducible coiled-coil 1; ULK, Unc-51-like kinase.

Metamorphic proteins at the basis of human autophagy initiation and lipid transfer.

Autophagy is a conserved intracellular degradation pathway that generates de novo double-membrane autophagosomes to target a wide range of material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length seven-subunit human autophagy initiation supercomplex built on a core complex of ATG13-101 and ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to switch between distinct folds. The slow spontaneous metamorphic conversion is rate limiting for the self-assembly of the supercomplex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.

Altered tRNA dynamics during translocation on slippery mRNA as determinant of spontaneous ribosome frameshifting.

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and -1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the -1-frame triplet, resulting in slippage followed by recruitment of the -1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.

A resourceful methodology to profile indolic auxins produced by rhizo-fungi using spectrophotometry and HPTLC.

Plant growth-promoting fungi play an important role in development of sustainable agriculture. In the current study, 13 fungal strains were isolated from the rhizosphere of healthy Triticum aestivum (wheat) plant and screened for their indolic auxin production potential. Aspergillus flavus strain PGFW, Aspergillus niger strain BFW and Aspergillus caespitosus strain DGFW were amongst the most efficient indolic auxin-producing strains. Indolic auxins such as indole 3 acetate (IAA), indole 3 butyrate (IBA) and indole 3 propionate (IPA) are produced by fungi. The conventional method to assess the IAA production is through a spectrophotometric assay using Salkowski's reagent, which quantifies all indolic auxins and not individual auxins. Moreover, it was also observed that the absorption maxima (λmax) of the samples, when compared to that of standard indole-3-acetic acid, showed deviation from the latter, indicative of production of a mixture of indolic derivatives by the fungi. Hence, for further profiling of these indolic compounds, high-performance thin layer chromatography (HPTLC) based protocol was standardized to precisely detect and quantify individual indolic auxins like IAA, IBA and IPA in the range of 100-1000 ng per spot. HPTLC analysis also showed that the fungal strains produce different indolic auxins in media with and without fortification of tryptophan, with the production of indolic auxins being enhanced in presence of tryptophan. Thus, this standardized HPTLC protocol is an efficient and sensitive methodology to separate and quantify the indolic derivatives.