Kotaro Tsuboyama

In macroautophagy, cytoplasmic contents are sequestered into the double-membrane autophagosome, which fuses with the lysosome to become the autolysosome. It has been thought that the autophagy-related (ATG) conjugation systems are required for autophagosome formation. Here, we found that autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 17-positive autophagosome-like structures could be generated even in the absence of the ATG conjugation systems, although at a reduced rate. These syntaxin 17-positive structures could further fuse with lysosomes, but degradation of the inner autophagosomal membrane was significantly delayed. Accordingly, autophagic activity in ATG conjugation-deficient cells was strongly suppressed. We suggest that the ATG conjugation systems, which are likely required for the closure (i.e., fission) of the autophagosomal edge, are not absolutely essential for autolysosome formation but are important for efficient degradation of the inner autophagosomal membrane.

Abstract Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale 1 . However, the energetics driving folding are invisible in these structures and remain largely unknown 2 . The hidden thermodynamics of folding can drive disease 3,4 , shape protein evolution 5–7 and guide protein engineering 8–10 , and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40–72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMass spectrometry of nucleic acid components. Trimethylsilyl derivatives of nucleotides, nucleosides, and basesJames A. McCloskey, A. M. Lawson, K. Tsuboyama, P. M. Krueger, and R. N. StillwellCite this: J. Am. Chem. Soc. 1968, 90, 15, 4182–4184Publication Date (Print):July 1, 1968Publication History Published online1 May 2002Published inissue 1 July 1968https://pubs.acs.org/doi/10.1021/ja01017a062https://doi.org/10.1021/ja01017a062research-articleACS PublicationsRequest reuse permissionsArticle Views372Altmetric-Citations117LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMass spectrometry of nucleic acid components. Trimethylsilyl derivatives of nucleotidesJames A. McCloskey, A. M. Lawson, Richard N. Stillwell, M. M. Tacker, and K. TsuboyamaCite this: J. Am. Chem. Soc. 1971, 93, 4, 1014–1023Publication Date (Print):February 1, 1971Publication History Published online1 May 2002Published inissue 1 February 1971https://pubs.acs.org/doi/10.1021/ja00733a039https://doi.org/10.1021/ja00733a039research-articleACS PublicationsRequest reuse permissionsArticle Views247Altmetric-Citations78LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts