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Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells.

Watson P, Townley AK, Koka P, Palmer KJ, Stephens DJ - Traffic (2006)

Bottom Line: Sec16 localizes to ERES independent of Sec23/24 and Sec13/31.Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts.Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.

ABSTRACT
The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.

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Sar1-GTP mediates assembly of Sec16 at ERESA) Cells expressing ECFP-Sar1-T39N (GDP restricted, blue in merge) were labelled with antibodies to detect endogenous Sec16 (green) and Sec24C (red). The majority of punctate Sec16 localization is lost; those few punctate spots of Sec16 that remain also label with anti-Sec24C antibodies, as they do in non-transfected cells. B) Cells were transfected with both ECFP-Sar1-T39N (blue in merge) and Venus-Sec16 (green) and immunolabelled for endogenous Sec24C (red). C) Cells transfected with ECFP-Sar1-H79G (blue in merge) were labelled for endogenous Sec16 (green) and Sec24C (red). Sec24C is not present on Sec16-positive structures (arrowheads) D) 3D imaging of paraformaldehyde-fixed cells expressing both ECFP-Sar1-H79G (green in merge) and Venus-Sec16 (red) shows that both proteins localize to large curved and round structures. E) 3D rendering of deconvolved stacks from cells expressing both ECFP-Sar1-H79G and Venus-Sec16 reveal that these objects often form near complete spheres. F) Immunolabelling of ECFP-Sar1-H79G (blue in merge) and Venus-Sec16 (green) labelled structures shows that they do not contain Sec24C (red). Boxed regions show threefold enlargements of selected areas. Representative images are shown from four independent experiments in which a total of 400 cells were inspected visually. All bars = 10 μm.
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fig07: Sar1-GTP mediates assembly of Sec16 at ERESA) Cells expressing ECFP-Sar1-T39N (GDP restricted, blue in merge) were labelled with antibodies to detect endogenous Sec16 (green) and Sec24C (red). The majority of punctate Sec16 localization is lost; those few punctate spots of Sec16 that remain also label with anti-Sec24C antibodies, as they do in non-transfected cells. B) Cells were transfected with both ECFP-Sar1-T39N (blue in merge) and Venus-Sec16 (green) and immunolabelled for endogenous Sec24C (red). C) Cells transfected with ECFP-Sar1-H79G (blue in merge) were labelled for endogenous Sec16 (green) and Sec24C (red). Sec24C is not present on Sec16-positive structures (arrowheads) D) 3D imaging of paraformaldehyde-fixed cells expressing both ECFP-Sar1-H79G (green in merge) and Venus-Sec16 (red) shows that both proteins localize to large curved and round structures. E) 3D rendering of deconvolved stacks from cells expressing both ECFP-Sar1-H79G and Venus-Sec16 reveal that these objects often form near complete spheres. F) Immunolabelling of ECFP-Sar1-H79G (blue in merge) and Venus-Sec16 (green) labelled structures shows that they do not contain Sec24C (red). Boxed regions show threefold enlargements of selected areas. Representative images are shown from four independent experiments in which a total of 400 cells were inspected visually. All bars = 10 μm.

Mentions: In S. cerevisiae, Sec16 is recruited to neutral liposomes in a Sar1- and GTP-dependent manner (17); however, in P. pastoris, expression of a GDP-restricted form of Sar1 does not result in a loss of Sec16 localization (18). Here, we found that in human cells, expression of Sar1-T39N [a GDP-restricted form (9)] resulted in a near complete loss of Sec16 and Sec24 from punctate structures (Figure 7A, also Sec31, not shown). Note that Sec16 and Sec24C co-localize in non-transfected cells (Figure 7A); in Sar1-T39N-transfected cells, those puncta that remain contain both Sec16 and Sec24C (albeit very faintly, Figure 7A, box), suggesting that they are indeed bona fide ERES. Quantification shows that >90% of Sec24 was lost from these sites (n = 200 ERES from four independent experiments). Expression of ECFP-Sar1-T39N together with Venus-Sec16 resulted in localization of Venus-Sec16 to punctate spots but displacement of >95% of Sec24C from the membrane [Figure 7B (n = 200 ERES from four independent experiments)]. Again the presence of very faint Sec24 localization to these sites strongly suggests that they are indeed bona fide ERES. These data suggest that, at endogenous levels of expression, Sec16 requires Sar1-GTP for association with ERES; however, when overexpressed, Venus-Sec16 can assemble at ERES independent of GTP-loading of Sar1. We interpret this as an inherent capacity of the protein to self-assemble that is exaggerated on overexpression. An alternative explanation is that the higher levels of Sec16 resulting from overexpression are able to bind to pre-existing ERES more efficiently. This result is in direct contrast to the situation in P. pastoris, where expression of Sar1-T34N does not cause a loss of Sec16 or Sec23/24 from ERES (18). While this might reflect a difference between these species, it could also be explained by differences in expression level. Previous work has shown that GTP is required with Sar1 to recruit S. cerevisiae Sec16 to dioleoylphosphatidylcholine/dioleolyphosphatidylethanolamine (DOPC/DOPE) liposomes (although Sec16 binds independently to other liposomes) (17). As Sec16 does not become recruited to the entire ER membrane (where Sar1 will become activated by Sec12), the simplest interpretation of our data is that Sar1-GTP is required to maintain the association of Sec16 with ERES, perhaps by preventing its dissociation. Overexpression of GFP-Sec16 overcomes this, such that ERES are maintained even in the presence of Sar1-T39N. These data further support the idea of self-assembly of higher order Sec16 structures.


Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells.

Watson P, Townley AK, Koka P, Palmer KJ, Stephens DJ - Traffic (2006)

Sar1-GTP mediates assembly of Sec16 at ERESA) Cells expressing ECFP-Sar1-T39N (GDP restricted, blue in merge) were labelled with antibodies to detect endogenous Sec16 (green) and Sec24C (red). The majority of punctate Sec16 localization is lost; those few punctate spots of Sec16 that remain also label with anti-Sec24C antibodies, as they do in non-transfected cells. B) Cells were transfected with both ECFP-Sar1-T39N (blue in merge) and Venus-Sec16 (green) and immunolabelled for endogenous Sec24C (red). C) Cells transfected with ECFP-Sar1-H79G (blue in merge) were labelled for endogenous Sec16 (green) and Sec24C (red). Sec24C is not present on Sec16-positive structures (arrowheads) D) 3D imaging of paraformaldehyde-fixed cells expressing both ECFP-Sar1-H79G (green in merge) and Venus-Sec16 (red) shows that both proteins localize to large curved and round structures. E) 3D rendering of deconvolved stacks from cells expressing both ECFP-Sar1-H79G and Venus-Sec16 reveal that these objects often form near complete spheres. F) Immunolabelling of ECFP-Sar1-H79G (blue in merge) and Venus-Sec16 (green) labelled structures shows that they do not contain Sec24C (red). Boxed regions show threefold enlargements of selected areas. Representative images are shown from four independent experiments in which a total of 400 cells were inspected visually. All bars = 10 μm.
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Related In: Results  -  Collection

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fig07: Sar1-GTP mediates assembly of Sec16 at ERESA) Cells expressing ECFP-Sar1-T39N (GDP restricted, blue in merge) were labelled with antibodies to detect endogenous Sec16 (green) and Sec24C (red). The majority of punctate Sec16 localization is lost; those few punctate spots of Sec16 that remain also label with anti-Sec24C antibodies, as they do in non-transfected cells. B) Cells were transfected with both ECFP-Sar1-T39N (blue in merge) and Venus-Sec16 (green) and immunolabelled for endogenous Sec24C (red). C) Cells transfected with ECFP-Sar1-H79G (blue in merge) were labelled for endogenous Sec16 (green) and Sec24C (red). Sec24C is not present on Sec16-positive structures (arrowheads) D) 3D imaging of paraformaldehyde-fixed cells expressing both ECFP-Sar1-H79G (green in merge) and Venus-Sec16 (red) shows that both proteins localize to large curved and round structures. E) 3D rendering of deconvolved stacks from cells expressing both ECFP-Sar1-H79G and Venus-Sec16 reveal that these objects often form near complete spheres. F) Immunolabelling of ECFP-Sar1-H79G (blue in merge) and Venus-Sec16 (green) labelled structures shows that they do not contain Sec24C (red). Boxed regions show threefold enlargements of selected areas. Representative images are shown from four independent experiments in which a total of 400 cells were inspected visually. All bars = 10 μm.
Mentions: In S. cerevisiae, Sec16 is recruited to neutral liposomes in a Sar1- and GTP-dependent manner (17); however, in P. pastoris, expression of a GDP-restricted form of Sar1 does not result in a loss of Sec16 localization (18). Here, we found that in human cells, expression of Sar1-T39N [a GDP-restricted form (9)] resulted in a near complete loss of Sec16 and Sec24 from punctate structures (Figure 7A, also Sec31, not shown). Note that Sec16 and Sec24C co-localize in non-transfected cells (Figure 7A); in Sar1-T39N-transfected cells, those puncta that remain contain both Sec16 and Sec24C (albeit very faintly, Figure 7A, box), suggesting that they are indeed bona fide ERES. Quantification shows that >90% of Sec24 was lost from these sites (n = 200 ERES from four independent experiments). Expression of ECFP-Sar1-T39N together with Venus-Sec16 resulted in localization of Venus-Sec16 to punctate spots but displacement of >95% of Sec24C from the membrane [Figure 7B (n = 200 ERES from four independent experiments)]. Again the presence of very faint Sec24 localization to these sites strongly suggests that they are indeed bona fide ERES. These data suggest that, at endogenous levels of expression, Sec16 requires Sar1-GTP for association with ERES; however, when overexpressed, Venus-Sec16 can assemble at ERES independent of GTP-loading of Sar1. We interpret this as an inherent capacity of the protein to self-assemble that is exaggerated on overexpression. An alternative explanation is that the higher levels of Sec16 resulting from overexpression are able to bind to pre-existing ERES more efficiently. This result is in direct contrast to the situation in P. pastoris, where expression of Sar1-T34N does not cause a loss of Sec16 or Sec23/24 from ERES (18). While this might reflect a difference between these species, it could also be explained by differences in expression level. Previous work has shown that GTP is required with Sar1 to recruit S. cerevisiae Sec16 to dioleoylphosphatidylcholine/dioleolyphosphatidylethanolamine (DOPC/DOPE) liposomes (although Sec16 binds independently to other liposomes) (17). As Sec16 does not become recruited to the entire ER membrane (where Sar1 will become activated by Sec12), the simplest interpretation of our data is that Sar1-GTP is required to maintain the association of Sec16 with ERES, perhaps by preventing its dissociation. Overexpression of GFP-Sec16 overcomes this, such that ERES are maintained even in the presence of Sar1-T39N. These data further support the idea of self-assembly of higher order Sec16 structures.

Bottom Line: Sec16 localizes to ERES independent of Sec23/24 and Sec13/31.Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts.Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.

ABSTRACT
The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.

Show MeSH
Related in: MedlinePlus