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Showing content from https://pubmed.ncbi.nlm.nih.gov/22358839/ below:

Ubiquitin-dependent regulation of COPII coat size and function

. 2012 Feb 22;482(7386):495-500. doi: 10.1038/nature10822. Ubiquitin-dependent regulation of COPII coat size and function

Affiliations

Affiliation

¹ Department of Molecular and Cell Biology, University of California at Berkeley, California 94720, USA.

PMID: 22358839
PMCID: PMC3292188
DOI: 10.1038/nature10822

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Ubiquitin-dependent regulation of COPII coat size and function

Lingyan Jin et al. Nature. 2012.

. 2012 Feb 22;482(7386):495-500. doi: 10.1038/nature10822. Affiliation

¹ Department of Molecular and Cell Biology, University of California at Berkeley, California 94720, USA.

PMID: 22358839
PMCID: PMC3292188
DOI: 10.1038/nature10822

Item in Clipboard

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.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures
Figure 1. Cul3 regulates mESC morphology

a.…

Figure 1. Cul3 regulates mESC morphology

a. Left: D3 mESCs were plated on gelatin and…

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…

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

a. D3 mESCs were…

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…

Figure 3. Cul3 ^Klhl12 monoubiquitinates Sec31

a. Immunoprecipitates of ^FLAG Klhl12 or ^FLAG Klhl9 were analyzed…

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…

a.…

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

a. Localization of induced ^FLAG Klhl12 (green) and Sec31…

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.…

Figure 5. Cul3 ^Klhl12 promotes collagen export

a. IMR90 cells transfected with ^FLAG Klhl12, ^FLAG Klhl12…

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).