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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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Depletion of Sec16 from human cells disrupts COPII assembly and ER-to-Golgi transportA) Depletion of Sec16 expression using siRNA. HeLa cells were transfected with siRNA duplexes targeting Sec16 [pool of four duplexes (Sec16 pool), lamin A/C, a duplex targeting the 3′ UTR of Sec16 (Sec16 3′ UTR) and a further Sec16 duplex targeting the coding region (Sec16 b)]. Cell lysates were then immunoblotted for Sec16 (250 kD), lamin A/C (84 kD) or GAPDH (as a loading control, 40 kD) as indicated. Note that the duplex termed Sec16b only depletes a subset of splice forms of Sec16. Cells depleted of Sec16 using a pool of four siRNA duplexes were methanol fixed and labelled with antibodies specific for B) Sec24C, C) Sec31A, D) ERGIC-53, E) β’-COP and F) giantin as indicated. Bar (all panels) = 10 μm.
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fig05: Depletion of Sec16 from human cells disrupts COPII assembly and ER-to-Golgi transportA) Depletion of Sec16 expression using siRNA. HeLa cells were transfected with siRNA duplexes targeting Sec16 [pool of four duplexes (Sec16 pool), lamin A/C, a duplex targeting the 3′ UTR of Sec16 (Sec16 3′ UTR) and a further Sec16 duplex targeting the coding region (Sec16 b)]. Cell lysates were then immunoblotted for Sec16 (250 kD), lamin A/C (84 kD) or GAPDH (as a loading control, 40 kD) as indicated. Note that the duplex termed Sec16b only depletes a subset of splice forms of Sec16. Cells depleted of Sec16 using a pool of four siRNA duplexes were methanol fixed and labelled with antibodies specific for B) Sec24C, C) Sec31A, D) ERGIC-53, E) β’-COP and F) giantin as indicated. Bar (all panels) = 10 μm.

Mentions: To determine the requirement for Sec16 in ERES organization and membrane traffic from the ER, we depleted the protein from cells using RNA interference. Immunoblotting with antibodies directed against Sec16 showed that we were able to efficiently deplete Sec16 with a pool of four duplexes as well as with individual duplexes (Figure 5A). One duplex depleted only one of the two bands observed, suggesting that it is not effective against all splice forms that are expressed in HeLa cells. The role of these different splice forms remains unclear. Depletion of lamin A/C was used as a control and none of the small interfering RNA (siRNA) duplexes used caused any changes in expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figure 5A). Using a pool of four siRNA duplexes targeting Sec16 (Sec16 smartPOOL), we found that depletion of Sec16 resulted in fewer punctate spots of Sec24C (Figure 5B) and Sec31A (Figure 5C) within the cell, particularly significant within the juxtanuclear area. Quantification of the number of spots in the periphery of cells shows a decrease from 25 ± 10/10 μm2 in lamin A/C-depleted cells to 4 ± 3/10 μm2 in Sec16-depleted cells (n = 100 cells from three independent experiments). Similarly, ERGIC-53 was seen to be localized to the ER (Figure 5D), fewer punctate spots of COPI were visible in the periphery (Figure 5E), and giantin localization was more dispersed and puncta compared with its typical ribbon-like appearance (Figure 5F). Ninety-six per cent of cells show these distributions following depletion of Sec16, likely a reflection of siRNA transfection (or depletion) efficiency. The same results were obtained when siRNA experiments were performed both with a pool of four duplexes as well as individual duplexes included in the pool and other independent duplexes, including the one targeting the 3′ untranslated region (UTR) of the Sec16 messenger RNA (see Materials and Methods).


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)

Depletion of Sec16 from human cells disrupts COPII assembly and ER-to-Golgi transportA) Depletion of Sec16 expression using siRNA. HeLa cells were transfected with siRNA duplexes targeting Sec16 [pool of four duplexes (Sec16 pool), lamin A/C, a duplex targeting the 3′ UTR of Sec16 (Sec16 3′ UTR) and a further Sec16 duplex targeting the coding region (Sec16 b)]. Cell lysates were then immunoblotted for Sec16 (250 kD), lamin A/C (84 kD) or GAPDH (as a loading control, 40 kD) as indicated. Note that the duplex termed Sec16b only depletes a subset of splice forms of Sec16. Cells depleted of Sec16 using a pool of four siRNA duplexes were methanol fixed and labelled with antibodies specific for B) Sec24C, C) Sec31A, D) ERGIC-53, E) β’-COP and F) giantin as indicated. Bar (all panels) = 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1761133&req=5

fig05: Depletion of Sec16 from human cells disrupts COPII assembly and ER-to-Golgi transportA) Depletion of Sec16 expression using siRNA. HeLa cells were transfected with siRNA duplexes targeting Sec16 [pool of four duplexes (Sec16 pool), lamin A/C, a duplex targeting the 3′ UTR of Sec16 (Sec16 3′ UTR) and a further Sec16 duplex targeting the coding region (Sec16 b)]. Cell lysates were then immunoblotted for Sec16 (250 kD), lamin A/C (84 kD) or GAPDH (as a loading control, 40 kD) as indicated. Note that the duplex termed Sec16b only depletes a subset of splice forms of Sec16. Cells depleted of Sec16 using a pool of four siRNA duplexes were methanol fixed and labelled with antibodies specific for B) Sec24C, C) Sec31A, D) ERGIC-53, E) β’-COP and F) giantin as indicated. Bar (all panels) = 10 μm.
Mentions: To determine the requirement for Sec16 in ERES organization and membrane traffic from the ER, we depleted the protein from cells using RNA interference. Immunoblotting with antibodies directed against Sec16 showed that we were able to efficiently deplete Sec16 with a pool of four duplexes as well as with individual duplexes (Figure 5A). One duplex depleted only one of the two bands observed, suggesting that it is not effective against all splice forms that are expressed in HeLa cells. The role of these different splice forms remains unclear. Depletion of lamin A/C was used as a control and none of the small interfering RNA (siRNA) duplexes used caused any changes in expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figure 5A). Using a pool of four siRNA duplexes targeting Sec16 (Sec16 smartPOOL), we found that depletion of Sec16 resulted in fewer punctate spots of Sec24C (Figure 5B) and Sec31A (Figure 5C) within the cell, particularly significant within the juxtanuclear area. Quantification of the number of spots in the periphery of cells shows a decrease from 25 ± 10/10 μm2 in lamin A/C-depleted cells to 4 ± 3/10 μm2 in Sec16-depleted cells (n = 100 cells from three independent experiments). Similarly, ERGIC-53 was seen to be localized to the ER (Figure 5D), fewer punctate spots of COPI were visible in the periphery (Figure 5E), and giantin localization was more dispersed and puncta compared with its typical ribbon-like appearance (Figure 5F). Ninety-six per cent of cells show these distributions following depletion of Sec16, likely a reflection of siRNA transfection (or depletion) efficiency. The same results were obtained when siRNA experiments were performed both with a pool of four duplexes as well as individual duplexes included in the pool and other independent duplexes, including the one targeting the 3′ untranslated region (UTR) of the Sec16 messenger RNA (see Materials and Methods).

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