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Development of cytosol-facing organelle outer membrane proximity labeling systems in Arabidopsis

ABSTRACT: The cytosol-facing organelle outer membrane (OM) is a layer that separates from but also communicates with cellular compartments for the import and exchange of proteins, metabolites and signaling molecules. OM resident proteins are also the targets of cellular surveillance systems by which the cell controls organelle homeostasis for promoting cell survival under stress. However, discovery of OM proteins and their dynamically interacting partners by traditional approaches can be very challenging. In this study, we developed an outer membrane proximity labeling system (OMPL) using biotin ligase mediated proximity biotinylation to map the proximity proteome of the cytosol-facing mitochondria, chloroplast and peroxisome outer membranes in living Arabidopsis cells. The power of this optimized system is highlighted by the discovery of the cytosolic factors and OM receptor candidates that are potentially involved in protein local translation and translocation, membrane contact sites and organelle quality control. Moreover, this OMPL plants generated in this study can be used for rapid and specific isolation of intact mitochondria and peroxisome. Our findings provide ample ground for further investigations on important questions in organelle biology.

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Xiaohong Zhu

PROVIDER: PXD039752 | JPOST Repository | Wed Jan 31 00:00:00 GMT 2024

REPOSITORIES: jPOST

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Project description:Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the posttranscriptional Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the post-transcriptional dynamics of mRNAs.

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Development of cytosol-facing organelle outer membrane proximity labeling systems in Arabidopsis

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