City centres of the apostle Paul

Thesis (MPhil)--Stellenbosch University, 2002On title page: Master of Philosophy (Bible Skills)ENGLISH ABSTRACT: The apostle Paul was called and commissioned to the city centres of the eastern Mediterranean world. These city centres were places of power, trade, wealth and travel. They were the nerve centres of civilisation in the East. People from various parts of the Roman Empire were found in these major cities. The city was therefore a highly significant institution in the Roman Empire. The governing authorities wanted hellenization and romanization to spread from these cities. The Christian leaders also decided that Christianity had to spread in the Roman Empire from its city centres. The apostle Paul's Christian mission was therefore to the various cities in the Roman Empire. The sociohistorical realities in these cities therefore formed the context of Paul's life and apostolic work and determined his relation to a city. The political, social, cultural and religious factors in a city could therefore impinge on his life and work. The apostle Paul was usually drawn to these large cities where he could find Jewish communities. As Christianity was resting on a Jewish foundation, his initial strategy was his work in the synagogues amongst the Jews. Paul also needed an alternative venue for his Christian work in the city. These alternate venues were usually the private homes of individuals who had become Christians. In these homes Paul established his church in a city. The hosts in these homes would usually become the benefactors and leaders in the church. Paul's apostolic work in a city was also done in the city streets. His church therefore became thoroughly mixed in terms of social status, however, the church gave all equal rights and privileges. When Paul left a city, he also placed on them the responsibility to reach their surrounding regions and provinces with the Christian message. These cities therefore had to be strategically located. The apostle Paul chose five specific cities that had an advantageous geographical position in the Roman provinces to complete his apostolic work in the eastern Mediterranean world.AFRIKAANSE OPSOMMING: Die apostel Paulus was geroep en opgedrag vir die stedelike sentrums van die ooste Middellandse wereld. Hierdie stedelike sentrums was plekke van invloedryke mag, ekonomiese handel, rykdom en reis aktiwitiete. Hulle was ook die kern van menslike beskawing in die Ooste. Bevolkings groepe vanuit verskeie dele van die Romeinse ryk was in hierdie groot stede te vinde. Stede was 'n hoogs betekensvolle instelling in die Romeinse ryk. Die politieke owerhede wou he dat hellenization en romanization moes sprei van hierdie stede. Die Christelike leiers het ook besluit dat Christendom moes in die stede van die Romeinse ryk sprei. Die apostel Paulus se Christelike sending was dus tot die verskillende stede in die Romeinse ryk. Die sosio-historiese realiteite in hierdie stede was die samehang van Paulus se apostoliese werk en het ook sy verhouding met die betrokke stede bepaal. Die politieke, maatskaplike, kulturele en godsdienstige faktore in 'n stad kon dus 'n invloed uitoefen op sy lewe en werk. Paulus was gewoonlik aangetrokke tot hierdie groot stede waar Joodse gemeenskappe te vinde was. Aangesien Christendom in die Joodse geloof gegrondves was, was sy aanvanklike strategie om sy werk te loots in sinagoge waar Joode te vinde was. Paulus het ook 'n alternatiewe ontmoetings plek vir sy Christelike werk in die stede nodig gehad. Hierdie alternatiewe ontmoetingsplekke was gewoonlik in die huise van indiwidue wat Christene geword het. Die eienaar van hierdie huishouding het gedien as gasheer, weldoener en leier in die kerk. Paulus het ook sy apostoliese werk voortgesit in die stedelike strate. Sy kerke het as gevolg hiervan 'n gemende samelewing status gehad, nogtans het hy gepoog om alle Christene gelykwaardig te stel. Wanneer Paulus 'n stad verlaat het, het hy het ook aan hulle die verantwoordelikheid gegee om uit te reik na hulomliggende streke en provinsies met die Christen boodskap. Hierdie stede moes dus strategies gelee wees. Paulus het vyf spesifieke stede wat 'n voordelig geologiese posisie in die Romeinse provinsies uitgeken om sy apostoliese werk te voltooi in die ooste Middellandse wereld

The lipid flippase Drs2 regulates anterograde transport of Atg9 during autophagy

Macroautophagy/autophagy is a conserved catabolic pathway during which cellular material is sequestered within newly formed double-membrane vesicles called autophagosomes and delivered to the lytic compartment of eukaryotic cells for degradation. Autophagosome biogenesis depends on the core autophagy-related (Atg) machinery, and involves a massive supply and remodelling of membranes. To gain insight into the lipid remodelling mechanisms during autophagy, we have systematically investigated whether lipid flippases are required for this pathway in the yeast Saccharomyces cerevisiae. We found that the flippase Drs2, which transfers phosphatidylserine and phosphatidylethanolamine from the lumenal to the cytosolic leaflet of the limiting membrane at the trans-Golgi network, is required for normal progression of autophagy. We also show that Drs2 is important for the trafficking of the core Atg protein Atg9. Atg9 is a transmembrane protein important for autophagosome biogenesis and its anterograde transport from its post-Golgi reservoirs to the site of autophagosome formation is severely impaired in the absence of Drs2. Thus, our results identify a novel autophagy player and highlight that membrane asymmetry regulates early autophagy steps. Abbreviations: ABs: autophagic bodies; Atg: autophagy-related; BiFC: bimolecular fluorescence microscopy; Cvt: cytoplasm-to-vacuole targeting; ER: endoplasmic reticulum; P4-ATPases: type IV P-type ATPases; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PS: phosphatidylserine; PtdIns3P: phosphatidylinositol-3-phosphate; TGN: trans-Golgi network; WT: wild typ

The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes

Lysosomes mediate degradation of macromolecules to their precursors for their cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. The Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein, and an effector of the minor Rab5 isoform Ypt52. Strikingly, the bph1▵ mutant has lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy inducing conditions. In agreement, proteomics of bph1▵ vacuoles reveal an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1▵ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases

­­Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy

During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9ʹs role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.</p

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.</p

Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion

Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8