Daniel Klionsky: A full plate for autophagy

Klionsky proves that those who can, teach as well as do

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein

A switch element in the autophagy E2 Atg3 mediates allosteric regulation across the lipidation cascade

Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12-Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3(E123IR) makes intramolecular interactions restraining Atg3's catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3's E123IR protects the E2 similar to UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy

Does relevancy matter?

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138329/1/bmb21052.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138329/2/bmb21052_am.pd

Neurodegeneration - Good riddance to bad rubbish

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62887/1/441819a.pd

Teaching the telephone book

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147825/1/bmb21193_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147825/2/bmb21193.pd

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and non-canonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Purification and biochemical characterization of the ATH1 gene product, vacuolar acid trehalase, from Saccharomyces cerevisiae

AbstractThe disaccharide trehalose plays a critical role in yeast cell survival during conditions of environmental stress. The vacuole of the yeast Saccharomyces cerevisiae contains an enzyme, acid trehalose (ATH), that is capable of degrading trehalose. Recently, a gene required for ATH activity, ATH1, was cloned and sequenced [Destruelle et al., (1995) Yeast 11, 1015–1025]. The relationship between ATH1 and ATH, however, was not determined. We have purified ATH and shown that it is the ATH1 gene product; peptide sequences from the purified protein correspond to the deduced amino acid sequence of Ath1p. In addition, antiserum to Ath1p specifically recognizes purified ATH

Wait, can you remind me just why we need another journal focused on autophagy?

Well, because you ask that question, we are going to attempt to explain exactly why we do indeed need another journal focused on autophagy. If you are reading this far, you presumably know what “autophagy” means, so we do not have to impress upon you the importance of this topic, and how autophagic dysfunction is associated with numerous diseases in humans (okay, we felt compelled to slip that in anyway). Nor do we think that you need to be introduced to the journal Autophagy, which is just starting its eighteenth year and publishes papers on pretty much any topic; at least any topic that is connected to autophagy, which, after all, means pretty much any topic, if you get our drift. So, if Autophagy has done so well and serves such an important purpose, why do we need another journal? To find the answer, read on