Protocols,Toolboxes and Resource papers

In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.     

Atg11

Selective macroautophagy uses double-membrane vesicles, termed autophagosomes, to transport cytoplasmic pathogens, organelles and protein complexes to the vacuole for degradation. Autophagosomes are formed de novo by membrane fusion events at the phagophore assembly site (PAS). Therefore, precursor membrane material must be targeted and transported to the PAS. While some autophagy-related (Atg) proteins, such as Atg9 and Atg11, are known to be involved in this process, most of the mechanistic details are not understood. Previous work has also implicated the small Rab-family GTPase Ypt1 in the process, identifying Trs85 as a unique subunit of the TRAPPIII targeting complex and showing that it plays a macroautophagy-specific role; however, the relationship between Ypt1, Atg9 and Atg11 was not clear. Now, a recent report shows that Atg11 is a Trs85-specific effector of the Rab Ypt1, and may act as a classic coiled-coil membrane tether that targets Atg9-containing membranes to the PAS. Here, we review this finding in the context of what is known about Atg11, other Rab-dependent coiled-coil tethers, and other tethering complexes involved in autophagosome formation.     

Osh6 overexpression extends the lifespan of yeast by increasing vacuole fusion

In yeast cells, the vacuole divides and fuses in each round of cell cycle. While mutants defective in vacuole fusion are “wild type” for vegetative growth, most have shortened replicative lifespans under caloric restriction (CR) condition, a manipulation that extends lifespan in wild type cells. To explore whether vacuole fusion extends lifespan, we screened for genes that can complement the fusion defect of selected mutants (erg6Δ, a sterol mutant; nyv1Δ, a mutant involved in the vacuolar SNARE complex and vac8Δ, a vacuolar membrane protein mutant). This screen revealed that Osh6, a member of the oxysterol-binding protein family, can complement the vacuole fusion defect of nyv1Δ, but not erg6Δ or vac8Δ, suggesting that Osh6’s function in vacuole fusion is partly dependent on membrane ergosterol and Vac8. To measure the effect of OSH6 on lifespan, we replaced the endogenous promoter of OSH6 with a shorter version of the ERG6 promoter to obtain P_ERG6-OSH6. This mutant construct significantly extended the replicative lifespan in a wild type background and in a nyv1Δ mutant. Interestingly, P_ERG6-OSH6 cells were more sensitive to drugs that inhibit the activity of the TOR complex 1 (TORC1) than wild type cells. Moreover, a P_ERG6-OSH6 tor1Δ double mutant demonstrated a greatly shortened lifespan, suggesting a genetic interaction between Osh6 and Tor1. Since active TORC1 stimulates vacuole scission and CR downregulates TORC1, Osh6 may link these two pathways by adjusting vacuolar membrane organization to extend lifespan.     

There is more to autophagy than induction

Considerable attention has been paid to the topic of autophagy induction. In part, this is because of the potential for modulating this process for therapeutic purposes. Of course we know that induced autophagy can also be problematic—for example, when trying to eliminate an established tumor that might be relying on autophagy for its own cytoprotective uses. Accordingly, inhibitory mechanisms have been considered; however, the corresponding studies have tended to focus on the pathways that block autophagy under noninducing conditions, such as when nutrients are available. In contrast, relatively little is known about the mechanisms for inhibiting autophagy under inducing conditions. Yet, this type of regulation must be occurring on a routine basis. We know that dysregulation of autophagy, e.g., due to improper activation of Beclin 1 leading to excessive autophagy activity, can cause cell death.¹ Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122:927 - 39; http://dx.doi.org/10.1016/j.cell.2005.07.002; PMID: 16179260 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Accordingly, we assume that during starvation or other inducing conditions there must be a mechanism to modulate autophagy. That is, once you turn it on, you do not want to let it continue unchecked. But how is autophagy downregulated when the inducing conditions still exist?     

Why just eat in,when you can also eat out?

The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).     

Rat β-glucuronidase as a reporter protein for the analysis of the plant secretory pathway

Abstract

E. coliβ-glucuronidase, a cytosolic enzyme, was found not to be a good reporter enzyme for secretion studies in plants. In this study, we chose to test and adapt an animal β-glucuronidase as a better reporter protein for the secretory pathway of plants. We modified rat β-glucuronidase to obtain secreted and vacuolar variants. Five different C-termini were produced: the original C-terminus of the rat enzyme, a 19 codon deletion (Δ19), a 15 codon deletion (Δ15) and fusions of the Δ19 or Δ15 termini with the last 6 or 7 codons of the vacuolar sorting determinant of tobacco chitinase A, respectively. The signal sequence of the rat β-glucuronidase polypeptide was replaced by the sequence encoding the signal peptide of tobacco chitinase A. In a transient expression system, the best enzymatic activity was found with β-glucuronidase having the 15 codons deletion, therefore Δ15 (secRGUS) and Δ15 + Chi (RGUS-Chi) were further evaluated and their efficiency of secretion or vacuolar targeting were tested under different conditions. To determine the correct targeting of reporter genes, we compared the localization of β-glucuronidase and of an endogenous marker, α-mannosidase. Treating cells with drugs that specifically affect different aspects of the secretory pathway also tested the validity of RGUS-based reporters. A non-specific inhibitor such as cytochalasin D and a wide range inhibitor such as BFA were compared with specific inhibitors such as wortmannin and bafilomycin A1. Finally, monensin and NH₄Cl were used to evaluate the role of vacuolar pH in correct RGUS-Chi targeting. The two new reporter proteins proved to be good tools for our studies in the transient expression system in tobacco protoplasts and for further applications.     

Old and New Synthetic Capacities of baker's Yeast

An overview on some recent developments in the use of Baker's yeast as a biocatalyst is given. Some recent examples concerning oxido-reductases, phosphatases and other less defined catalytic activities are discussed.     

Pyruvate Decarboxylase Catalysed Formation of Terpenoid α-hydroxy Ketones

Enzyme extracts of the wild type yeast Zygosaccharomyces bisporus were applied for the pyruvate decarboxylase catalysed condensation of pyruvate and (R)-(+)-and (S)-(?)-perillyl aldehyde, (±)-citronellal, neral, geranial or (R)-(?)-myrtenal to form novel α-hydroxy ketones. Best yields were obtained when the transformation medium contained 25% (v/v) of the cosolvent N,N-dimethylformamide. Conversion of (R)-(+)-perillyl aldehyde to (1R)-1-hydroxy-1-[(4’R)-4’-isopropenyl-1-cyclohexen-1-yl]-2-propanone proceeded highly stereospecifically (>99% de), whereas the stereoselectivity was somewhat less in the transformation of (S)-(?)-perillyl aldehyde (58% de) and (R)-(?)-myrtenal (92% de). All of the new compounds imparted characteristic odour impressions as determined by means of GC-olfactometry.     

Protection of yeast lacking the Ure2 protein against the toxicity of heavy metals and hydroperoxides by antioxidants

The aim of this study was to examine the protection of the yeast lacking the “antioxidant-like” prion precursor protein (Ure2p), by antioxidants and to elucidate how modification of redox homeostasis affects toxicity of agents inducing oxidative stress in the Δure2 cells. We found a diverse ability of a range of antioxidants to ameliorate the hypersensitivity of the Δure2 disruptant to oxidants and heavy metal ions. Glutathione and then ascorbate were the most effective antioxidants; Tempol, Trolox and melatonin were much less effective or even hampered the growth of the Δure2 cells exposed to tested agents. The intracellular level of ROS was augmented in the Δure2 mutant under normal growth conditions (1.7-fold), and after treatment with H₂O₂ (2.3-fold) and Cd(II) (2.8-fold), with respect to its wild-type counterpart. Glutathione was unable to prevent the increase in ROS production caused by CdCl₂. The Δure2 disruptant was also hypersensitive to heat shock, like mutants lacking glutathione S-transferases.