Limits...
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

Protein Network Associated with the RING Domain of RNF26(A) Workflow scheme for the identification and validation of proteins interacting with the cytosolic domain of RNF26. RING-associated RNF26 proteome consists of membrane-associated adaptor proteins and a DUB USP15 (for proteomic analysis details, see Figure S4A). Localization of RNF26-interacting proteins EPS15, TOLLIP, TAX1BP1, SQSTM1, and USP15 are depicted schematically (for representative marker overlays see Figure S4A). CCV, clathrin-coated vesicle; EE, early endosome; RE, recycling endosome; LE, late endosome; Ly, lysosome; TGN, trans-Golgi network; AUT, autophagosome; PM, plasma membrane.(B) Intracellular distribution (fractional distance analysis, mean shown in red) of LEs (CD63) and TGN (TGN46) in MelJuSo cells as a function of indicated siRNA perturbations. For cell shape analysis, see Figure S4B. For combinatorial silencing of vesicle adaptors, see Figure S4C.(C) Representative z-cross section (3D) image overlays of CD63 (green, upper panels) or TGN46 (green, bottom panels) with nuclear DAPI (blue) in MelJuSo cells are shown with the corresponding protein levels of silenced targets (left lanes) as compared to the control (right lanes).(D) Effect of GFP-TOLLIP (green) overexpression on the organization and dynamics of acidified vesicles (LTVs, magenta) in HeLa cells. Left panels: representative single confocal plane fluorescence image overlays taken at the start of the time lapse. Right panels: corresponding vesicle displacement rates (blue, immobile; red, max mobility) observed during the 343-s time interval; zoom-ins highlight boxed PN regions. Quantification of LTV dynamics (displacement/s relative to untransfected cells) as a function of TOLLIP is shown above the images; n = 2. See also Figure S4D and Movies S5A and S5B.(E) Top graph: quantification (Mander’s overlap) of SR101 entry into acidified vesicles (LTVs) as a function of time (min) in control MelJuSo cells (siC; control dataset in common with Figure 2C) versus those depleted of TOLLIP (siTOLLIP). n = 2. Bottom graph: total uptake of SR101 in control or TOLLIP-depleted MelJuSo cells as measured by flow cytometry, expressed as fold increase normalized to t = 0 as a function of time; n = 3. Scale bars, 10 μm.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4930482&req=5

fig4: Protein Network Associated with the RING Domain of RNF26(A) Workflow scheme for the identification and validation of proteins interacting with the cytosolic domain of RNF26. RING-associated RNF26 proteome consists of membrane-associated adaptor proteins and a DUB USP15 (for proteomic analysis details, see Figure S4A). Localization of RNF26-interacting proteins EPS15, TOLLIP, TAX1BP1, SQSTM1, and USP15 are depicted schematically (for representative marker overlays see Figure S4A). CCV, clathrin-coated vesicle; EE, early endosome; RE, recycling endosome; LE, late endosome; Ly, lysosome; TGN, trans-Golgi network; AUT, autophagosome; PM, plasma membrane.(B) Intracellular distribution (fractional distance analysis, mean shown in red) of LEs (CD63) and TGN (TGN46) in MelJuSo cells as a function of indicated siRNA perturbations. For cell shape analysis, see Figure S4B. For combinatorial silencing of vesicle adaptors, see Figure S4C.(C) Representative z-cross section (3D) image overlays of CD63 (green, upper panels) or TGN46 (green, bottom panels) with nuclear DAPI (blue) in MelJuSo cells are shown with the corresponding protein levels of silenced targets (left lanes) as compared to the control (right lanes).(D) Effect of GFP-TOLLIP (green) overexpression on the organization and dynamics of acidified vesicles (LTVs, magenta) in HeLa cells. Left panels: representative single confocal plane fluorescence image overlays taken at the start of the time lapse. Right panels: corresponding vesicle displacement rates (blue, immobile; red, max mobility) observed during the 343-s time interval; zoom-ins highlight boxed PN regions. Quantification of LTV dynamics (displacement/s relative to untransfected cells) as a function of TOLLIP is shown above the images; n = 2. See also Figure S4D and Movies S5A and S5B.(E) Top graph: quantification (Mander’s overlap) of SR101 entry into acidified vesicles (LTVs) as a function of time (min) in control MelJuSo cells (siC; control dataset in common with Figure 2C) versus those depleted of TOLLIP (siTOLLIP). n = 2. Bottom graph: total uptake of SR101 in control or TOLLIP-depleted MelJuSo cells as measured by flow cytometry, expressed as fold increase normalized to t = 0 as a function of time; n = 3. Scale bars, 10 μm.

Mentions: To understand how an ER-located protein exerts control over the endosomal system and the TGN, we sought out interacting partners of the cytosolic tail of RNF26. Mass spectrometric analysis of proteins co-precipitating with either GST-ΔTM or GST-RING (Figures 4A and S4A) identified three membrane-associated adaptor proteins functioning in sorting and trafficking of endo- or exocytic vesicles—EPS15 (Benmerah et al., 1999), T6BP/TAX1BP1 (Morriswood et al., 2007), and TOLLIP (Ankem et al., 2011), a ubiquitin scaffold p62/SQSTM1 (Ciani et al., 2003) known for its role in autophagy (Lippai and Löw, 2014) and a DUB USP15, which localizes to the nucleus and cytosol, targeting the transforming growth factor β (TGF-β) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathways (Eichhorn et al., 2012, Schweitzer et al., 2007). Collectively, cargo specificities of the three former proteins afford broad coverage of both endocytic and biosynthetic vesicle trajectories (Figures 4A and S4A), implying that by association with different vesicle-targeting adaptors, RNF26 may influence positioning of a wide range of endosomes and the TGN. Silencing the above proteins (excluding USP15) produced marked LE dispersion (Figures 4B and 4C). By contrast, TGN vesicle dispersion resulted only from depletion of the TGN-associated adaptor TAX1BP1 and SQSTM1, but not of endocytic adaptors EPS15 and TOLLIP (Figures 4B and 4C), and overall cell shape parameters were profoundly altered only by depletion of TAX1BP1 (Figure S4B). Further, co-silencing multiple adaptors resulted in additive effects on CD63 distribution (Figure S4C), underscoring the contribution of multi-directional traffic to the global architecture of the LE compartment.


An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport
Protein Network Associated with the RING Domain of RNF26(A) Workflow scheme for the identification and validation of proteins interacting with the cytosolic domain of RNF26. RING-associated RNF26 proteome consists of membrane-associated adaptor proteins and a DUB USP15 (for proteomic analysis details, see Figure S4A). Localization of RNF26-interacting proteins EPS15, TOLLIP, TAX1BP1, SQSTM1, and USP15 are depicted schematically (for representative marker overlays see Figure S4A). CCV, clathrin-coated vesicle; EE, early endosome; RE, recycling endosome; LE, late endosome; Ly, lysosome; TGN, trans-Golgi network; AUT, autophagosome; PM, plasma membrane.(B) Intracellular distribution (fractional distance analysis, mean shown in red) of LEs (CD63) and TGN (TGN46) in MelJuSo cells as a function of indicated siRNA perturbations. For cell shape analysis, see Figure S4B. For combinatorial silencing of vesicle adaptors, see Figure S4C.(C) Representative z-cross section (3D) image overlays of CD63 (green, upper panels) or TGN46 (green, bottom panels) with nuclear DAPI (blue) in MelJuSo cells are shown with the corresponding protein levels of silenced targets (left lanes) as compared to the control (right lanes).(D) Effect of GFP-TOLLIP (green) overexpression on the organization and dynamics of acidified vesicles (LTVs, magenta) in HeLa cells. Left panels: representative single confocal plane fluorescence image overlays taken at the start of the time lapse. Right panels: corresponding vesicle displacement rates (blue, immobile; red, max mobility) observed during the 343-s time interval; zoom-ins highlight boxed PN regions. Quantification of LTV dynamics (displacement/s relative to untransfected cells) as a function of TOLLIP is shown above the images; n = 2. See also Figure S4D and Movies S5A and S5B.(E) Top graph: quantification (Mander’s overlap) of SR101 entry into acidified vesicles (LTVs) as a function of time (min) in control MelJuSo cells (siC; control dataset in common with Figure 2C) versus those depleted of TOLLIP (siTOLLIP). n = 2. Bottom graph: total uptake of SR101 in control or TOLLIP-depleted MelJuSo cells as measured by flow cytometry, expressed as fold increase normalized to t = 0 as a function of time; n = 3. Scale bars, 10 μm.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930482&req=5

fig4: Protein Network Associated with the RING Domain of RNF26(A) Workflow scheme for the identification and validation of proteins interacting with the cytosolic domain of RNF26. RING-associated RNF26 proteome consists of membrane-associated adaptor proteins and a DUB USP15 (for proteomic analysis details, see Figure S4A). Localization of RNF26-interacting proteins EPS15, TOLLIP, TAX1BP1, SQSTM1, and USP15 are depicted schematically (for representative marker overlays see Figure S4A). CCV, clathrin-coated vesicle; EE, early endosome; RE, recycling endosome; LE, late endosome; Ly, lysosome; TGN, trans-Golgi network; AUT, autophagosome; PM, plasma membrane.(B) Intracellular distribution (fractional distance analysis, mean shown in red) of LEs (CD63) and TGN (TGN46) in MelJuSo cells as a function of indicated siRNA perturbations. For cell shape analysis, see Figure S4B. For combinatorial silencing of vesicle adaptors, see Figure S4C.(C) Representative z-cross section (3D) image overlays of CD63 (green, upper panels) or TGN46 (green, bottom panels) with nuclear DAPI (blue) in MelJuSo cells are shown with the corresponding protein levels of silenced targets (left lanes) as compared to the control (right lanes).(D) Effect of GFP-TOLLIP (green) overexpression on the organization and dynamics of acidified vesicles (LTVs, magenta) in HeLa cells. Left panels: representative single confocal plane fluorescence image overlays taken at the start of the time lapse. Right panels: corresponding vesicle displacement rates (blue, immobile; red, max mobility) observed during the 343-s time interval; zoom-ins highlight boxed PN regions. Quantification of LTV dynamics (displacement/s relative to untransfected cells) as a function of TOLLIP is shown above the images; n = 2. See also Figure S4D and Movies S5A and S5B.(E) Top graph: quantification (Mander’s overlap) of SR101 entry into acidified vesicles (LTVs) as a function of time (min) in control MelJuSo cells (siC; control dataset in common with Figure 2C) versus those depleted of TOLLIP (siTOLLIP). n = 2. Bottom graph: total uptake of SR101 in control or TOLLIP-depleted MelJuSo cells as measured by flow cytometry, expressed as fold increase normalized to t = 0 as a function of time; n = 3. Scale bars, 10 μm.
Mentions: To understand how an ER-located protein exerts control over the endosomal system and the TGN, we sought out interacting partners of the cytosolic tail of RNF26. Mass spectrometric analysis of proteins co-precipitating with either GST-ΔTM or GST-RING (Figures 4A and S4A) identified three membrane-associated adaptor proteins functioning in sorting and trafficking of endo- or exocytic vesicles—EPS15 (Benmerah et al., 1999), T6BP/TAX1BP1 (Morriswood et al., 2007), and TOLLIP (Ankem et al., 2011), a ubiquitin scaffold p62/SQSTM1 (Ciani et al., 2003) known for its role in autophagy (Lippai and Löw, 2014) and a DUB USP15, which localizes to the nucleus and cytosol, targeting the transforming growth factor β (TGF-β) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathways (Eichhorn et al., 2012, Schweitzer et al., 2007). Collectively, cargo specificities of the three former proteins afford broad coverage of both endocytic and biosynthetic vesicle trajectories (Figures 4A and S4A), implying that by association with different vesicle-targeting adaptors, RNF26 may influence positioning of a wide range of endosomes and the TGN. Silencing the above proteins (excluding USP15) produced marked LE dispersion (Figures 4B and 4C). By contrast, TGN vesicle dispersion resulted only from depletion of the TGN-associated adaptor TAX1BP1 and SQSTM1, but not of endocytic adaptors EPS15 and TOLLIP (Figures 4B and 4C), and overall cell shape parameters were profoundly altered only by depletion of TAX1BP1 (Figure S4B). Further, co-silencing multiple adaptors resulted in additive effects on CD63 distribution (Figure S4C), underscoring the contribution of multi-directional traffic to the global architecture of the LE compartment.

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