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An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport

View Article: PubMed Central - PubMed

ABSTRACT

Through a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

No MeSH data available.


Related in: MedlinePlus

RNF26/SQSTM1 Complex Positions and Retains Adaptor-Selected Vesicles(A) Overlay zooms of frames selected from a time lapse (Movie S7) of vesicles marked by GFP-TOLLIP (green) in the presence of TRQ-SQSTM1 (blue) and RFP-RNF26 (red) in HeLa cells. Arrowheads point to two vesicles profiled in (B). Scale bar, 2.5 μm.(B) Plots of signal intensities over time corresponding to a positioned vesicle 1 (top graph) and a released vesicle 2 (bottom graph) as observed in (A). Dashed lines show background signal for each channel.(C) Quantification of adaptor-selected vesicle dynamics (mobile, white; positioned, black) expressed as % of vesicles per category (number counted given above each bar). GFP-marked vesicles (green); vesicles colocalizing with RFP-RNF26/-ΔRING and/or TRQ-SQSTM1 (white and cyan, respectively). See also Figures S6 and S7 and Movies S6A and S6B.(D) Co-localization (Mander’s overlap) between RNF26 and GFP-TOLLIP in control (siC) and SQSTM1-depleted (siSQSTM1) MelJuSo cells.(E) Model of vesicle positioning in the PN cloud by the RNF26 system. (1) Adaptor-selected (green) vesicles are subject to fast microtubule-based transport when unanchored by RNF26. (2) Catalytically competent RNF26 (light red) recruits SQSTM1 (blue) and mediates ubiquitin ligation (red), which serves to attract UBDs of specific vesicle-associated adaptors. On engagement, this multi-protein complex positions cognate vesicles (early, recycling, and late endosomes, and TGN) in the perinuclear space. (3) Dissociation of the RNF26/SQSTM1 complex, promoted by the DUB USP15 (yellow), releases target vesicles for (4) fast transport into the cell periphery.
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fig7: RNF26/SQSTM1 Complex Positions and Retains Adaptor-Selected Vesicles(A) Overlay zooms of frames selected from a time lapse (Movie S7) of vesicles marked by GFP-TOLLIP (green) in the presence of TRQ-SQSTM1 (blue) and RFP-RNF26 (red) in HeLa cells. Arrowheads point to two vesicles profiled in (B). Scale bar, 2.5 μm.(B) Plots of signal intensities over time corresponding to a positioned vesicle 1 (top graph) and a released vesicle 2 (bottom graph) as observed in (A). Dashed lines show background signal for each channel.(C) Quantification of adaptor-selected vesicle dynamics (mobile, white; positioned, black) expressed as % of vesicles per category (number counted given above each bar). GFP-marked vesicles (green); vesicles colocalizing with RFP-RNF26/-ΔRING and/or TRQ-SQSTM1 (white and cyan, respectively). See also Figures S6 and S7 and Movies S6A and S6B.(D) Co-localization (Mander’s overlap) between RNF26 and GFP-TOLLIP in control (siC) and SQSTM1-depleted (siSQSTM1) MelJuSo cells.(E) Model of vesicle positioning in the PN cloud by the RNF26 system. (1) Adaptor-selected (green) vesicles are subject to fast microtubule-based transport when unanchored by RNF26. (2) Catalytically competent RNF26 (light red) recruits SQSTM1 (blue) and mediates ubiquitin ligation (red), which serves to attract UBDs of specific vesicle-associated adaptors. On engagement, this multi-protein complex positions cognate vesicles (early, recycling, and late endosomes, and TGN) in the perinuclear space. (3) Dissociation of the RNF26/SQSTM1 complex, promoted by the DUB USP15 (yellow), releases target vesicles for (4) fast transport into the cell periphery.

Mentions: To test whether SQSTM1 dictates endosome positioning at RNF26, we monitored vesicle dynamics via specific membrane adaptors in living cells co-expressing fluorescent SQSTM1. We observed stable PN contacts between GFP-adaptors (“green”) and RFP-RNF26 (“red”) that were overwhelmingly positive for TRQ-SQSTM1 (“blue”; Figure S7A; Movie S6A), with tripartite complex formation (appearing “white” in the overlay) strongly correlated to fixed positional residence of vesicles marked by EPS15, TAX1BP1, and TOLLIP (Figures 7A–7C). By contrast, the vast majority of only GFP-positive “green” vesicles remained subject to fast transport (Figure 7C; Movie S6A). As expected, RNF26 lacking its RING domain could not mediate stable contacts with TRQ-SQSTM1 and failed to stabilize vesicles in position over time (Figures 7C and S7A; Movie S6B). Similar to TRQ-SQSTM1, TRQ-ubiquitin was found at contracts between GFP-TOLLIP-positive vesicles retained by RFP-RNF26 (but not RFP-ΔRING), while highly mobile vesicles were free of ligase and ubiquitin contacts (Figures S7B and 7C).


An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport
RNF26/SQSTM1 Complex Positions and Retains Adaptor-Selected Vesicles(A) Overlay zooms of frames selected from a time lapse (Movie S7) of vesicles marked by GFP-TOLLIP (green) in the presence of TRQ-SQSTM1 (blue) and RFP-RNF26 (red) in HeLa cells. Arrowheads point to two vesicles profiled in (B). Scale bar, 2.5 μm.(B) Plots of signal intensities over time corresponding to a positioned vesicle 1 (top graph) and a released vesicle 2 (bottom graph) as observed in (A). Dashed lines show background signal for each channel.(C) Quantification of adaptor-selected vesicle dynamics (mobile, white; positioned, black) expressed as % of vesicles per category (number counted given above each bar). GFP-marked vesicles (green); vesicles colocalizing with RFP-RNF26/-ΔRING and/or TRQ-SQSTM1 (white and cyan, respectively). See also Figures S6 and S7 and Movies S6A and S6B.(D) Co-localization (Mander’s overlap) between RNF26 and GFP-TOLLIP in control (siC) and SQSTM1-depleted (siSQSTM1) MelJuSo cells.(E) Model of vesicle positioning in the PN cloud by the RNF26 system. (1) Adaptor-selected (green) vesicles are subject to fast microtubule-based transport when unanchored by RNF26. (2) Catalytically competent RNF26 (light red) recruits SQSTM1 (blue) and mediates ubiquitin ligation (red), which serves to attract UBDs of specific vesicle-associated adaptors. On engagement, this multi-protein complex positions cognate vesicles (early, recycling, and late endosomes, and TGN) in the perinuclear space. (3) Dissociation of the RNF26/SQSTM1 complex, promoted by the DUB USP15 (yellow), releases target vesicles for (4) fast transport into the cell periphery.
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fig7: RNF26/SQSTM1 Complex Positions and Retains Adaptor-Selected Vesicles(A) Overlay zooms of frames selected from a time lapse (Movie S7) of vesicles marked by GFP-TOLLIP (green) in the presence of TRQ-SQSTM1 (blue) and RFP-RNF26 (red) in HeLa cells. Arrowheads point to two vesicles profiled in (B). Scale bar, 2.5 μm.(B) Plots of signal intensities over time corresponding to a positioned vesicle 1 (top graph) and a released vesicle 2 (bottom graph) as observed in (A). Dashed lines show background signal for each channel.(C) Quantification of adaptor-selected vesicle dynamics (mobile, white; positioned, black) expressed as % of vesicles per category (number counted given above each bar). GFP-marked vesicles (green); vesicles colocalizing with RFP-RNF26/-ΔRING and/or TRQ-SQSTM1 (white and cyan, respectively). See also Figures S6 and S7 and Movies S6A and S6B.(D) Co-localization (Mander’s overlap) between RNF26 and GFP-TOLLIP in control (siC) and SQSTM1-depleted (siSQSTM1) MelJuSo cells.(E) Model of vesicle positioning in the PN cloud by the RNF26 system. (1) Adaptor-selected (green) vesicles are subject to fast microtubule-based transport when unanchored by RNF26. (2) Catalytically competent RNF26 (light red) recruits SQSTM1 (blue) and mediates ubiquitin ligation (red), which serves to attract UBDs of specific vesicle-associated adaptors. On engagement, this multi-protein complex positions cognate vesicles (early, recycling, and late endosomes, and TGN) in the perinuclear space. (3) Dissociation of the RNF26/SQSTM1 complex, promoted by the DUB USP15 (yellow), releases target vesicles for (4) fast transport into the cell periphery.
Mentions: To test whether SQSTM1 dictates endosome positioning at RNF26, we monitored vesicle dynamics via specific membrane adaptors in living cells co-expressing fluorescent SQSTM1. We observed stable PN contacts between GFP-adaptors (“green”) and RFP-RNF26 (“red”) that were overwhelmingly positive for TRQ-SQSTM1 (“blue”; Figure S7A; Movie S6A), with tripartite complex formation (appearing “white” in the overlay) strongly correlated to fixed positional residence of vesicles marked by EPS15, TAX1BP1, and TOLLIP (Figures 7A–7C). By contrast, the vast majority of only GFP-positive “green” vesicles remained subject to fast transport (Figure 7C; Movie S6A). As expected, RNF26 lacking its RING domain could not mediate stable contacts with TRQ-SQSTM1 and failed to stabilize vesicles in position over time (Figures 7C and S7A; Movie S6B). Similar to TRQ-SQSTM1, TRQ-ubiquitin was found at contracts between GFP-TOLLIP-positive vesicles retained by RFP-RNF26 (but not RFP-ΔRING), while highly mobile vesicles were free of ligase and ubiquitin contacts (Figures S7B and 7C).

View Article: PubMed Central - PubMed

ABSTRACT

Through a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

No MeSH data available.


Related in: MedlinePlus