Dataset Information

AP-3 vesicles retain their coat and depend on the HOPS subunit Vps41 for tethering to vacuoles

ABSTRACT: Identification of Age2 as part of AP-3 vesicles using Turbo-ID

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Florian Fröhlich

LAB HEAD: Christian Ungermann

PROVIDER: PXD020623 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering.

Schoppe Jannis J Mari Muriel M Yavavli Erdal E Auffarth Kathrin K Cabrera Margarita M Walter Stefan S Fröhlich Florian F Ungermann Christian C

The EMBO journal 20200825 20

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To u ...[more]

PMID: 32840906

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Project description:One of the key issues affecting yields of biovanillin in cell factories such as Escherichia coli is product toxicity, the mechanisms of which are poorly understood. To identify targets for engineering improved strains, we have studied mechanisms of vanillin toxicity in E. coli using a two-pronged apparoach: (i) a global proteomic analysis supported by multiple physiological experiments and mutant analyses (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome sequencing. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin. Upregulated proteins included enzymes capable of converting aldehydes to potentially less toxic compounds; pentose phosphate pathways enzymes, fumarase C and enzymes of the glyoxylate shunt; proteins involved in several key stress responses, in particular oxidative stress; proteins mediating uptake and processing of metal ions. 1H-NMR confirmed that E. coli detoxifies vanillin by reduction to vanillyl alcohol; the aldehyde reductases YqhD and DkgA were highly upregulated by vanillin and the purified enzymes reduced vanillinin an NADPH dependent manner. Vanillin caused accumulation of reactive oxygen species and activated an oxidative stress response through SoxRS, OxyR and MarA pathways. Slow vanillin dependent copper (II) to copper (I) reduction lead to upregulation of the copA gene and growth in the presence of vanillin was hypersensitive to inhibition by copper ions. RT-PCR and mutant growth data suggested AcrD and AaeAB as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct non-synonymous SNPs in the citrate synthase gene gltA, in addition to strain specific mutations in cpdA, rob and marC. One strain had a large ~10 kb deletion including the marRAB region. Purifed variant GltA enzymes all showed higher activity due to a lowered Km for oxaloacetate. Our data provide new understanding and novel gene targets for engineering vanillin-tolerant strains of E. coli, including deletion of the efflux pumps eamA, acrA, and acrB, enhancing oxidative stress defences and NADPH production, improving copper homeostasis and increasing citrate synthase activity using variant enzymes.

Dataset Information

AP-3 vesicles retain their coat and depend on the HOPS subunit Vps41 for tethering to vacuoles

Publications

AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering.

Similar Datasets

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