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The biogenesis of double-membrane vesicles called autophagosomes, which sequester and transport intracellular material for degradation in lysosomes or vacuoles, is a central event in autophagy. This process requires a unique set of factors called autophagy-related (Atg) proteins. The Atg proteins assemble to organize the preautophagosomal structure (PAS), at which a cup-shaped membrane, the isolation membrane (or phagophore), forms and expands to become the autophagosome. The molecular mechanism of autophagosome biogenesis remains poorly understood. Previous studies have shown that Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 and localizes to the PAS to initiate autophagosome biogenesis; however, the molecular function of Atg2 remains unknown. In this study, we show that Atg2 has two membrane-binding domains in the N- and C-terminal regions and acts as a membrane tether during autophagosome formation in the budding yeast Saccharomyces cerevisiae. An amphipathic helix in the C-terminal region binds to membranes and facilitates Atg18 binding to PI3P to target the Atg2-Atg18 complex to the PAS. The N-terminal region of Atg2 is also involved in the membrane binding of this protein but is dispensable for the PAS targeting of the Atg2-Atg18 complex. Our data suggest that this region associates with the endoplasmic reticulum (ER) and is responsible for the formation of the isolation membrane at the PAS. Based on these results, we propose that the Atg2-Atg18 complex tethers the PAS to the ER to initiate membrane expansion during autophagosome formation.

A new isolate, Gordonia sp. strain TY-5, is capable of growth on propane and n-alkanes with C 13 to C 22 carbon chains as the sole source of carbon. In whole-cell reactions, significant propane oxidation to 2-propanol was detected. A gene cluster designated prmABCD, which encodes the components of a putative dinucleariron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced. A mutant with prmB disrupted (prmB::Kan r ) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane. These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium. Downstream of the prm genes, an open reading frame (adh1) encoding an NAD ؉ -dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized. The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan r ) indicate that Adh1 plays a major role in propane metabolism. Two additional NAD ؉ -dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation. On the basis of these results, we conclude that Gordonia sp. strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol.Gaseous n-alkanes ranging from C 2 to C 5 are recognized as components of nonmethane hydrocarbons, and the increased concentrations of these gases in the atmosphere threaten to destabilize ecosystems through a variety of mechanisms (48). Although these gases are produced as natural intermediates of bacterial, plant, and mammalian metabolism, the main sources of pollution are natural oil seepage and oil spills (42). From a biotechnological perspective, gaseous alkanes are inexpensive carbon sources for microbial cultivation, and the enzymes participating in the oxidation pathway promise to be versatile biocatalysts.A number of microorganisms have been isolated for their ability to use gaseous n-alkanes as a sole carbon source. In the case of bacteria, these abilities have been found in some Pseudomonas strains (57) and many strains belonging to the order Actinomycetales, such as those of the genera Rhodococcus, Mycobacterium, Corynebacterium, Nocardia, and Pseudonocardia (3,15). Some of the bacteria are known to degrade various environmental pollutants (trichloroethylene, chloroform, methyl ethers, etc.) through cometabolism with gaseous alkanes (13, 52).The pathways for the oxidation of gaseous alkanes have received little attention compared with those for the microbial oxidation of methane (34) and liquid n-alkanes (24). Recently, the terminal oxidation pathway of butane (butane 3 1-butanol 3 butyraldehyde 3 butyrate) by "Pseudomonas butanovora" has been confirmed through enzymological and genetic approaches (2, 14). The first reaction is catalyzed by a soluble butane m...

In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C 3 to C 10 ) and cyclic ketones (C 4 to C 8 ). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane 3 2-propanol 3 acetone 3 methyl acetate 3 acetic acid ؉ methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.Gordonia sp. strain TY-5 is an actinomycete that is capable of aerobic growth on gaseous propane as a carbon and energy source. Our previous study showed that propane is oxidized to 2-propanol by monooxygenase-mediated subterminal oxidation and then 2-propanol is further metabolized to acetone by three distinct NAD ϩ -dependent secondary alcohol dehydrogenases (27). Although n-alkanes can be metabolized to the corresponding ketones through subterminal oxidation in some bacteria, microbial metabolism of the downstream ketone is poorly understood (1,2,22,36,46,47). A variety of bacteria, including Gordonia sp. strain TY-5, are able to utilize acetone as a source of carbon and energy. Previous studies on bacterial acetone metabolism both in vivo and in vitro suggested that acetone can be metabolized in two ways. In most aerobic bacteria, acetone was hydroxylated by an O 2 -dependent reaction producing acetol (hydroxyacetone), although the corresponding enzyme system is not known (13,29,42,45). For most anaerobes (and some aerobes), acetone undergoes CO 2 -dependent carboxylation, yielding acetoacetate. Recently, the enzyme responsible for the latter reaction, acetone carboxylase (EC 6.4.1.6), has been purified and characterized (3,12,15,(39)(40)(41).This study was conducted to characterize acetone metabolism in propane-utilizing Gordonia sp. strain TY-5 at the enzymatic and gene levels. We first identified two acetone-induced proteins and cloned their corresponding genes, acmA and acmB. Subsequently, we showed that acetone is oxidized to methyl acetate by a novel Baeyer-Villiger monooxygenase (BVMO) (acmA gene product) and that the methyl acetate produced was hydrol...