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Retromer Endosome Exit Domains Serve Multiple Trafficking Destinations and Regulate Local G Protein Activation by GPCRs.

ABSTRACT: Retromer mediates sequence-directed cargo exit from endosomes to support both endosome-to-Golgi (retrograde transport) and endosome-to-plasma membrane (recycling) itineraries. It is not known whether these trafficking functions require cargos to exit endosomes separately via distinct transport intermediates or whether the same retromer-coated carriers can support both itineraries. We addressed this question by comparing human Wntless (Wls) and ?2 adrenergic receptor (?2AR), which require retromer physiologically for retrograde transport and recycling, respectively. We show here by direct visualization in living cells that both cargos transit primarily the same endosomes and exit via shared transport vesicles generated from a retromer-coated endosome domain. While both Wls and ?2AR clearly localize to the same retromer-coated endosome domains, Wls is consistently enriched more strongly. This enrichment difference is determined by distinct motifs present in the cytoplasmic tail of each cargo, with Wls using tandem ?-X-[L/M] motifs and ?2AR using a PDZ motif. Exchanging these determinants reverses the enrichment phenotype of each cargo but does not change cargo itinerary, verifying the multifunctional nature of retromer and implying that additional sorting must occur downstream. Quantitative differences in the degree of cargo enrichment instead underlie a form of kinetic sorting that impacts the rate of cargo delivery via both itineraries and determines the ability of ?2AR to activate its cognate G protein transducer locally from endosomes. We propose that mammalian retromer forms a multifunctional membrane coat that supports shared cargo exit for divergent trafficking itineraries and regulates signaling from endosomes.

SUBMITTER: Varandas KC

PROVIDER: S-EPMC5140749 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Retromer Endosome Exit Domains Serve Multiple Trafficking Destinations and Regulate Local G Protein Activation by GPCRs.

Varandas Katherine C KC Irannejad Roshanak R von Zastrow Mark M

Current biology : CB 20161110 23

Retromer mediates sequence-directed cargo exit from endosomes to support both endosome-to-Golgi (retrograde transport) and endosome-to-plasma membrane (recycling) itineraries. It is not known whether these trafficking functions require cargos to exit endosomes separately via distinct transport intermediates or whether the same retromer-coated carriers can support both itineraries. We addressed this question by comparing human Wntless (Wls) and β2 adrenergic receptor (β2AR), which require retrome ...[more]

PMID: 27839977

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Project description:The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β' and σ subunits, a core domain (CD) that contacts the RNAP β' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RPo stabilization in vitro, while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.IMPORTANCEMycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

Dataset Information

Retromer Endosome Exit Domains Serve Multiple Trafficking Destinations and Regulate Local G Protein Activation by GPCRs.

Publications

Retromer Endosome Exit Domains Serve Multiple Trafficking Destinations and Regulate Local G Protein Activation by GPCRs.

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