Ubiquitin-dependent regulation of COPII coat size and function

Abstract

Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII vesicles are approximately 60-80 nm in diameter, yet some must increase their size to accommodate 300-400 nm procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained elusive. Here, we identified the ubiquitin ligase CUL3-KLHL12 as a regulator of COPII coat formation. CUL3-KLHL12 catalyses the monoubiquitylation of the COPII-component SEC31 and drives the assembly of large COPII coats. As a result, ubiquitylation by CUL3-KLHL12 is essential for collagen export, yet less important for the transport of small cargo. We conclude that monoubiquitylation controls the size and function of a vesicle coat.

Figure 1. Cul3 regulates mESC morphology

a. Left: D3 mESCs were plated on gelatin and transfected with siRNAs targeting Cul3, which resulted in cell clustering (phase microscopy; upper panel) and compaction (confocal microscopy: vinculin, green; actin, red; DNA, blue). Right: Depletion of Cul3 from mouse 3T3 fibroblasts did not cause cell compaction. b. Cul3 is required for integrin-localization to the mESC plasma membrane. D3 mESCs were plated on gelatin (top two rows), growth-factor depleted matrigel, or collagen-IV. Following Cul3-depletion, cell compaction and integrin-targeting to the plasma membrane were analyzed by confocal microscopy (actin, red; β1-integrin, green; DNA, blue).

Figure 2. Klhl12 is a substrate adaptor for Cul3 in mESCs

a. D3 mESCs were subjected to differentiation, and mRNA-levels of indicated proteins were measured by qRT-PCR. b. Klhl12 protein is downregulated upon differentiation, as observed by immunoblot of above samples. c. Klhl12 is a critical Cul3-adaptor in mESCs. D3 mESCs were sensitized towards altered integrin-signaling with dasatinib and monitored for compaction by phase (upper panel) or confocal microscopy (actin, red; vinculin, green; DNA, blue).

Figure 3. Cul3^Klhl12 monoubiquitinates Sec31

a. Immunoprecipitates of ^FLAGKlhl12 or ^FLAGKlhl9 were analyzed by Silver staining and mass spectrometry. Asterisk: non-specific band; double asterisk: breakdown product of Klhl12. b. Sec13 was immunoprecipitated from HeLa cell lysates, and Sec31 and Klhl12 were detected by immunoblot. c. Klhl12 co-localizes with COPII, as seen by confocal microscopy (Klhl12, green; Sec13, red; DNA, blue). d. D3 mESCs grown on gelatin and depleted of Sec13 were analyzed for compaction by phase (top) or confocal microscopy (actin, red; vinculin, green; DNA, blue). e. Cul3^Klhl12 monoubiquitinates Sec31. Cul3^Nedd8-Rbx1 was incubated with Klhl12, Sec13/31, and ubiquitin or ^Hisubiquitin. f. In vitro ubiquitination of Sec31 by Cul3^Klhl12 or Cul3^{Klhl12-FG289AA} was performed as above. g. Sec31 is monoubiquitinated in vivo. Upper panels: Ubiquitin conjugates were purified under denaturing conditions from MG132-treated 293T cells expressing ^Hisubiquitin, ^HASec31, Klhl12, Cul3, or dominant-negative Cul3, and analyzed by αSec31-Western. Lower panels: the same experiment was performed with lysine-free ^Hisubiquitin, which only allowed Sec31-monoubiquitination on at least two sites (Sec31^ubi1 and Sec31^ubi*). h. Ubiquitin conjugates were purified from 293T cells expressing Klhl12 or Sec31-binding deficient Klhl12-mutants. i. Cul3 is essential for Sec31-ubiquitination in vivo. 293T cells were transfected with ^Hisubiquitin and siRNAs, and ubiquitin conjugates were analyzed for Sec31 by Western.

Figure 4. Cul3^Klhl12-dependent monoubiquitination enlarges COPII-structures

a. Localization of induced ^FLAGKlhl12 (green) and Sec31 (red) in 293T cells, monitored by confocal microscopy. Scale bar: 3μm. b. Klhl12-expressing HeLa cells were analyzed for Klhl12 (green) and Sec31, Sec13, or Sec24C (red) by confocal microscopy. Scale bar: 3μm. c. COPII-structures in HeLa cells transfected with ^FLAGKlhl12, lysine-free ubiquitin, or Cul3-siRNA, analyzed by confocal microscopy. Scale bar: 500nm. d. Upper panel: Thin-section EM of Klhl12-expressing or control HeLa cells (red arrow: Klhl12-dependent structures; blue arrow: small control vesicles). Scale bar: 500nm. Lower panel: Immunogold-EM of Klhl12 in transiently transfected HeLa (left) or stable 293T cells (right). Scale bar: 200nm. e. HeLa cells transfected with ^FLAGKlhl12, lysine-free ubiquitin, ^FLAGKlhl12^FG289AA, ^FLAGCul3^1-250, or ^FLAGCul3, were analyzed for localization of Klhl12/Cul3 (green) and Sec31 (red) by confocal microscopy. Scale bar: 5μm.

Figure 5. Cul3^Klhl12 promotes collagen export

a. IMR90 cells transfected with ^FLAGKlhl12, ^FLAGKlhl12^FG289AA, or ^FLAGKeap1 were analyzed by confocal microscopy (BTB, green; collagen-I, red; DNA, blue). When noted, cells were treated with chloroquine, MG132, brefeldin A (BFA), or dialyzed medium lacking ascorbate. Errors: standard deviation, n=3. b. Cell lysate (L) or culture medium (M) of IMR90 cells transfected with ^FLAGKlhl12, or ^FLAGKlhl12^FG289AA was analyzed by immunoblotting. c. Collagen-I localization was analyzed IMR90 cells expressing Klhl12, after re-addition of ascorbate. d. HT1080 cells stably expressing collagen-I were transfected with shRNAs against Cul3 and analyzed by confocal microscopy (transfection control GFP, green; PDI, blue; collagen-I, red). Error bars: standard deviation, n=3. e. D3 mESCs were treated with control siRNAs or siRNAs targeting Cul3 or Sec13 and analyzed by confocal microscopy (collagen-IV, green; actin, red; DNA, blue).