Protein folding state-dependent sorting at the Golgi apparatus

Abstract

In eukaryotic cells, organelle-specific protein quality control (PQC) is critical for maintaining cellular homeostasis. Despite the Golgi apparatus being the major protein processing and sorting site within the secretory pathway, how it contributes to PQC has remained largely unknown. Using different chemical biology-based protein unfolding systems, we reveal the segregation of unfolded proteins from folded proteins in the Golgi. Quality control (QC) substrates are subsequently exported in distinct carriers, which likely contain unfolded proteins as well as highly oligomerized cargo that mimic protein aggregates. At an additional sorting step, oligomerized proteins are committed to lysosomal degradation, while unfolded proteins localize to the endoplasmic reticulum (ER) and associate with chaperones. These results highlight the existence of checkpoints at which QC substrates are selected for Golgi export and lysosomal degradation. Our data also suggest that the steady-state ER localization of misfolded proteins, observed for several disease-causing mutants, may have different origins.

FIGURE 1:

Degradation profiles of Golgi-localized model QC substrates. (A) Domain outline of Golgi QC substrates targeted to the cis- (MAN2A1-EGFP-HT2) and trans- (ST6GAL1- and B4GT-EGFP-HT2) Golgi. (B) Flow cytometry analysis quantifying the levels of MAN2A1-EGFP-HT2 in HEK293 cells on the indicated treatments for 6 h (EPX, epoxomicin; BafA1, bafilomycin A). CHX was added 1 h prior to the indicated treatments. (C) Same analysis as in B for HEK293 cells expressing B4GT-EGFP-HT2 or ST6GAL1-EGFP-HT2 (n = 2, data represent mean ± SEM). (D) Flow cytometry analysis quantifying the levels of B4GT-EGFP-HT2 in HeLa cells on the indicated treatments for 6 h in the absence (left) or presence (right) of CHX (n = 3, data represent mean ± SEM, results of t test are shown).

FIGURE 2:

Formation of Golgi QC carriers containing Golgi QC model substrates. (A) Left panel: representative confocal microscopy images of HeLa cells expressing B4GT-SNAP-HT2 labeled with a fluorescent SNAP ligand at time 0 and incubated with DMSO control or HyT36 for 1 h at 37°C. Right panel: particles per cell were quantified in FIJI (n = 19 for DMSO, n = 22 for HyT36). (B) Left panel: representative confocal microscopy images of HeLa cells expressing B4GT-EGFP-DHFR* incubated for 4 h at 20°C followed by 1 h at 37°C with the stabilizer TMP or TMP washed out and treated with DMSO. Right panel: Particles per cell were quantified in FIJI (n = 24 for TMP and n = 27 for DMSO). (C) B4GT-EGFP-HT2–expressing HeLa cells were transfected with B4GT-RFP and treated with HyT36 for 4 h at 20°C followed by 1 h at 37°C. (D) B4GT-EGFP-HT2–expressing HeLa cells were treated with HyT36 and a HT TMR ligand for 4 h at 20°C followed by 1 h at 37°C. All images are maximum projections of z-stacks. Scale bars correspond to 10 µm. Bar graphs represent mean ± SEM, p values (t test) are shown.

FIGURE 3:

Unfolded proteins are segregated from folded proteins at the Golgi. (A) Stills from spinning disk confocal imaging of HeLa cells expressing B4GT-EGFP-HT2 that were treated with HyT36 or the HyT36(-Cl) control. After incubation at 20°C, cells were transferred to the microscope and recording started 40 min thereafter for 70 min at 37°C. White arrowheads indicate individual carriers containing B4GT-EGFP-HT2. Images are maximum projections of z-stacks at 41 and 65 min at 37°C. (B) Stills from LLSM studies. After incubation at 20°C, cells were imaged at 37°C. Arrows indicate the separation of the B4GT-GFP and B4GT-SNAP-HT2 signal during a 30-min imaging period. (C) Zoom-in images of stills of LLSM covering the time frame of 30 to 60 min at 37°C. The white arrow indicates a carrier containing B4GT-SNAP-HT2 forming at the Golgi and pinching off. Images are three-dimensional projections rendered in ClearVolume.

FIGURE 4:

Unfolded and aggregated Golgi QC substrates colocalize. (A) B4GT-SNAP-HT2 (labeled with TMR, magenta) expressing HeLa cells were transfected with B4GT-3xFM*-GFP (green). Cells were treated with HyT36 in the presence or absence of D/D solubilizer for 4 h at 20°C followed by 1 h 37°C. (B) Quantification of GFP-positive B4GT-SNAP-HT2 carriers per cell. (-D/D: n = 24, + D/D: n = 19). (C) Quantification of soluble B4GT-3xFM*-GFP (left panel) and B4GT-SNAP-HT2 (right panel) based on Western blot analysis. Raw data are shown in Supplemental Figure S4 (n = 2, data represent mean ± SD, results from t test are shown).

FIGURE 5:

QC carriers are targeted to the endo/lysosomal system. B4GT-SNAP-HT2 expressing HeLa cells were transfected with mCherry-RAB5A Q79L. Single slices and maximum projections of z-stacks for cells treated for 1 h at 37°C with DMSO (A) or HyT36 (B) are shown. Cartoon illustrates the individual slices of the z-stack (a and b). B4GT-SNAP-HT2 (labeled with Oregon Green), which is found in mCherry-RAB5A Q79L positive structures (magenta), is highlighted with white arrows. Cartoon illustrates the individual slices of the z-stack (a and b).

FIGURE 6:

Golgi QC substrates localize to the ER. (A) Time course analysis B4GT-SNAP-HT2 protein localization in HeLa cells on labeling with a TMR-SNAP ligand at time 0 and treated with HyT36 for the indicated times. (B) Colocalization of B4GT-SNAP-HT2 labeled with a TMR-SNAP ligand and treated with HyT36 for 4 h at 37°C with the ER marker BiP visualized by immunofluorescence using an Alexa Fluor 488–conjugated secondary antibody. All images are maximum projections of z-stacks. Scale bars correspond to 10 µm. (C) B4GT-EGFP-HT2–expressing HeLa cells were treated with DMSO or HyT36 for 4 h. The CV and integrated intensity of the GFP signal per cell were quantified. GFP intensity was normalized to DMSO treatment (n = 16, results from t test are shown) (D) GFP-trap pull down of B4GT-EGFP-HT2 from HeLa cells on treatment with HyT36 or DMSO control for 4 h at 37°C. Cells were treated with CHX 1 h prior and during the indicated treatments. Western blot analysis and quantification (data represent mean ± SEM) of association with ER chaperones are shown.

FIGURE 7:

Model of Golgi QC mechanisms: 1) unfolded proteins are separated from folded proteins at the Golgi; 2) subsequently, QC substrates are exported from the Golgi and targeted to the endo/lysosomal pathway; 3) at an additional QC checkpoint, aggregated proteins are committed to degradation at the lysosome. Unfolded Golgi proteins localize to the ER and associate with chaperones.