In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two A series of site-specific somatic DNA rearrangements is required for the assembly of the immunoglobulin and T-cell receptor (TCR) genes of the vertebrate immune system (for reviews, see Lewis 1994;Gellert 1997;Oettinger 1999). These V(D)J recombination events are initiated by the combined action of two lymphoid-specific proteins, RAG1 and RAG2. Together, these proteins recognize recombination signal sequences (RSSs) that flank immunoglobulin and TCR gene segments and introduce a double-strand break (DSB) at the border of the coding gene segment and the RSS. The RSS is comprised of conserved heptamer and nonamer elements separated by a spacer region of conserved length (12 or 23 bp) but variable sequence. The DSB is generated in two steps (McBlane et al. 1995). In the first step, a nick is introduced at the 5Ј end of the heptamer at the signal/coding boundary. In the second step, the newly liberated hydroxyl on the coding DNA attacks the phosphodiester bond of the opposing strand to form a hairpinned coding end and a blunt signal end. Subsequent processing and rejoining of the cleaved ends to form the mature coding segment and a signal junction requires the action of a number of proteins, including several involved in the repair of X-rayinduced damage (Jeggo 1998).The reactions carried out by the RAG proteins are chemically similar to those carried out by the transposase proteins of bacterial transposons such as Mu or Tn10 or by the integrase proteins of retroviruses such as HIV (Plasterk 1998;Roth and Craig 1998). In addition to carrying out the two steps of V(D)J cleavage, the RAG proteins can mediate transpositional recombination in vitro, joining the 3Ј OH of a cleaved signal end to an unrelated target sequence (Agrawal et al. 1998;. A shared feature of the strand transfer reactions of these mobile elements and the RAG recombinase is that they occur in the absence of an external energy source via a one-step transesterification reaction with no protein-DNA intermediate (van Gent et al. 1996). One distinction between the RAG proteins and other recombinases is the reversal of the initial cleavage site relative to the DNA recognition sequence. Specifically, RAG proteins nick the top strand (the 5Ј end of the heptamer at the signal/coding boundary), whereas MuA and Tn10 transposases, for...

The subunit dimerizes DNA polymerase III via interaction with the ␣ subunit, allowing DNA polymerase III holoenzyme to synthesize both leading and lagging strands simultaneously at the DNA replication fork. Here, we report a general method to map the limits of domains required for heterologous protein-protein interactions using surface plasmon resonance. The method employs fusion of a short biotinylation sequence at either the NH 2 or COOH terminus of the protein to be immobilized on streptavidin-derivatized biosensor chips. Inclusion of a hexahistidine sequence permits rapid purification and separation of the fusion protein from the endogenous Escherichia coli biotin carboxyl carrier protein. Ten deletions of the ␣ subunit were constructed and purified by Ni 2؉ -nitrilotriacetic acid chromatography and, when required, monomeric avidin chromatography. Each ␣ deletion protein was captured by streptavidin immobilized on a Pharmacia Biosensor BIAcore chip, and the binding activity of each ␣ deletion was analyzed using surface plasmon resonance. The subunit bound very tightly to a full-length amino-terminal fusion of the biotinylation sequence with ␣ (K D ϳ 70 pM). Four additional NH 2 -terminal ␣ deletion proteins (60, 240, 360, and 542 residues deleted) retained strong binding activity to the subunit (K D ‫؍‬ 0.19 -0.39 nM), whereas deletion of 705 residues or more from the NH 2 terminus of the ␣ subunit abolished binding activity. Full-length ␣ that contained a carboxyl-terminal fusion with the biotinylation sequence bound strongly (K D ‫؍‬ 0.37 nM). However, deletion of 48 amino acids from the COOH terminus totally eliminated binding. These results indicate that the COOH-terminal half of the ␣ subunit is involved in interaction. DNA polymerase III holoenzyme (holoenzyme)1 is the major DNA polymerase responsible for Escherichia coli chromosomal DNA synthesis. Holoenzyme consists of 10 individual subunits: ␣, ⑀, , , ␥, ␦, ␦Ј, , , and ␤ (McHenry, 1988;Kornberg, 1988), all of which act cooperatively in the coordinate and processive synthesis of leading and lagging strands (Wu et al., 1992a(Wu et al., , 1992bFay et al., 1982). Each subunit is encoded by a separate gene on the chromosome, except for and ␥, which are both expressed from dnaX. The subunit (71 kDa) is the full-length product of the dnaX gene; the ␥ subunit (47 kDa) is synthesized by a Ϫ1 translational frameshift and comprises the NH 2 -terminal two-thirds of the subunit (McHenry et al., 1989;Tsuchihashi and Kornberg, 1990;Blinkowa and Walker, 1990;Flower and McHenry, 1990).Polymerase III core (pol III) is composed of three different subunits: ␣, ⑀, and (McHenry and Crow, 1979). The ␣ subunit (dnaE product), a 130-kDa polypeptide, has intrinsic DNA polymerase activity (Welch and McHenry, 1982;Maki et al., 1985), the ⑀ subunit (dnaQ product) contains 3Ј 3 5Ј exonuclease activity for proofreading (Scheuermann and Echols, 1984), and the function of the subunit (holE product) is unknown (Carter et al., 1993;Studwell-Vaughan et al., 1993;Slater et al., 1994). ...

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V(D)J recombination occurs in two well-defined stages. In the first stage, a double-strand break (DSB) is introduced between the RSS and the flanking coding sequence (20,21,26). The resulting cleavage intermediates, a covalently closed hairpinned coding end and a blunt signal end, have been observed both in vivo (20, 21, 26) and in vitro (16, 30). In the second stage, the broken signal ends are ligated head-to-head to form signal joints, and the coding ends are sealed together in coding joints. Formation of the initial DSB requires the combined action of the two lymphoid-specific recombinase proteins RAG1 and RAG2 (16,17,25). No breaks are observed in cells lacking either one of these proteins (26). Subsequent resolution of these broken molecules relies on a series of steps that have yet to be defined, but which appear to involve a number of general DNA repair factors (11).The initial cleavage steps can be reproduced by using purified RAG1 and RAG2 proteins and an oligonucleotide substrate containing an RSS (16). Together the RAG proteins specifically recognize the RSS and introduce a DSB between the RSS and flanking coding sequence. Cleavage occurs in two steps (16). In the first step, a nick is introduced at the signal/ coding border on the top strand (see Fig. 1, single signal). In a second, separable step, the free 3Ј hydroxyl carries out a nucleophilic attack on the phosphodiester bond on the bottom strand, leaving a hairpinned coding flank and a blunt, 5Ј phosphorylated signal end, the same intermediates observed in vivo. Throughout this paper, "cleavage" refers to this hairpin and DSB formation.Efficient recombination requires a pair of RSSs, one 12-signal and one 23-signal (13). Because all immunoglobulin and T-cell receptor gene segments of a given type are flanked by signals of the same spacer length, this 12/23 rule serves to ensure that rearrangements occur only between elements that could create a functional receptor gene. While there is a requirement for a pair of signals for cleavage in vivo (28), in vitro conditions can be manipulated to permit a comparison of the requirements for cleavage at a single site versus cleavage at a signal pair (coupled cleavage). Single-site or coupled cleavage depends on the divalent metal ion (6, 32). With a single site, Mn 2ϩ permits hairpin formation, whereas only nicking proceeds in Mg 2ϩ . However, hairpin formation does occur in Mg 2ϩ when two signals are present (Fig. 1). Thus, cleavage is coupled in Mg 2ϩ , requiring two signals for efficient DSB formation.The requirement for two signals is separable in vitro from the requirement for a proper 12/23 pair. When cleavage is carried out in Mg 2ϩ with purified RAG1 and RAG2 proteins, the preference for a 12/23 pair over a 12/12 pair is only three-to fivefold (24,32). In vivo, the preference is closer to 100-fold (9, 28). In vitro cleavage in the presence of nuclear extract more readily approximates this preference, which has led to the suggestion that additional factors may be required to enforce the full extent o...

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubuleassociated protein 1 light chain 3 b) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvationinduced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/ threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.