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Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

ABSTRACT: Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.

SUBMITTER: Morales Quinones M

PROVIDER: S-EPMC3323037 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

Morales Quinones Mariana M Winston Jared T JT Stromhaug Per E PE

The Journal of biological chemistry 20111128 13

Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for b ...[more]

PMID: 22123825

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Project description:BackgroundThe Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.MethodsBy using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.ResultsThe high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.ConclusionsThe PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

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Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

Publications

Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

Similar Datasets

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