Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of ER-to-Golgi transport following overexpression of CFP-Sec16Cells expressing high levels of CFP-Sec16 were infected with Adenovirus to express tsO45-G-YFP at 39.5°C. Cells were then shifted to 32°C for A) 30 min or B) 90 min. A) In cells expressing a low level of CFP-Sec16, tsO45-G-YFP accumulates in the Golgi apparatus 30 min following shift to 32°C, while in cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER. B) Cells incubated at 32°C for 90 min were paraformaldehyde fixed and immunolabelled to detect tsO45-G-YFP that reaches the PM. In cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER (total) and does not reach the PM. C and D) Cells expressing GFP-Sec16 (asterisks) do not have significantly disrupted Golgi apparatus as determined by giantin localization (right hand panels). Bar (all panels) = 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1761133&req=5

fig04: Inhibition of ER-to-Golgi transport following overexpression of CFP-Sec16Cells expressing high levels of CFP-Sec16 were infected with Adenovirus to express tsO45-G-YFP at 39.5°C. Cells were then shifted to 32°C for A) 30 min or B) 90 min. A) In cells expressing a low level of CFP-Sec16, tsO45-G-YFP accumulates in the Golgi apparatus 30 min following shift to 32°C, while in cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER. B) Cells incubated at 32°C for 90 min were paraformaldehyde fixed and immunolabelled to detect tsO45-G-YFP that reaches the PM. In cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER (total) and does not reach the PM. C and D) Cells expressing GFP-Sec16 (asterisks) do not have significantly disrupted Golgi apparatus as determined by giantin localization (right hand panels). Bar (all panels) = 10 μm.

Mentions: To quantitatively monitor transport of secretory cargo, we used the model cargo protein tsO45-G-YFP, which can be accumulated in the ER at 39.5°C and released as a relatively synchronous wave of cargo transport at 32°C (22,23). In cells overexpressing enhanced cyan fluorescent protein (ECFP)-Sec16 at high level, export of tsO45-G-YFP from the ER and transport to the Golgi apparatus (Figure 4A, asterisk, 30 min after shift to the permissive temperature) was completely inhibited. Note that the adjacent cell in Figure 4A expressing a low level of CFP-Sec16 (localizing exclusively to ERES) shows no inhibition of transport to the Golgi apparatus. Further transport of tsO45-G-YFP to the plasma membrane (PM) (measured 90 min after shift to the permissive temperature) was completely inhibited by high expression of CFP-Sec16 (Figure 4B). In cells expressing these high levels of GFP-Sec16, the Golgi apparatus remains intact, indicating a block in ER export rather than simply a disrupted Golgi apparatus (Figure 4C, D).


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)

Inhibition of ER-to-Golgi transport following overexpression of CFP-Sec16Cells expressing high levels of CFP-Sec16 were infected with Adenovirus to express tsO45-G-YFP at 39.5°C. Cells were then shifted to 32°C for A) 30 min or B) 90 min. A) In cells expressing a low level of CFP-Sec16, tsO45-G-YFP accumulates in the Golgi apparatus 30 min following shift to 32°C, while in cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER. B) Cells incubated at 32°C for 90 min were paraformaldehyde fixed and immunolabelled to detect tsO45-G-YFP that reaches the PM. In cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER (total) and does not reach the PM. C and D) Cells expressing GFP-Sec16 (asterisks) do not have significantly disrupted Golgi apparatus as determined by giantin localization (right hand panels). Bar (all panels) = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1761133&req=5

fig04: Inhibition of ER-to-Golgi transport following overexpression of CFP-Sec16Cells expressing high levels of CFP-Sec16 were infected with Adenovirus to express tsO45-G-YFP at 39.5°C. Cells were then shifted to 32°C for A) 30 min or B) 90 min. A) In cells expressing a low level of CFP-Sec16, tsO45-G-YFP accumulates in the Golgi apparatus 30 min following shift to 32°C, while in cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER. B) Cells incubated at 32°C for 90 min were paraformaldehyde fixed and immunolabelled to detect tsO45-G-YFP that reaches the PM. In cells expressing a high level of CFP-Sec16 (asterisk), tsO45-G-YFP remains within the ER (total) and does not reach the PM. C and D) Cells expressing GFP-Sec16 (asterisks) do not have significantly disrupted Golgi apparatus as determined by giantin localization (right hand panels). Bar (all panels) = 10 μm.
Mentions: To quantitatively monitor transport of secretory cargo, we used the model cargo protein tsO45-G-YFP, which can be accumulated in the ER at 39.5°C and released as a relatively synchronous wave of cargo transport at 32°C (22,23). In cells overexpressing enhanced cyan fluorescent protein (ECFP)-Sec16 at high level, export of tsO45-G-YFP from the ER and transport to the Golgi apparatus (Figure 4A, asterisk, 30 min after shift to the permissive temperature) was completely inhibited. Note that the adjacent cell in Figure 4A expressing a low level of CFP-Sec16 (localizing exclusively to ERES) shows no inhibition of transport to the Golgi apparatus. Further transport of tsO45-G-YFP to the plasma membrane (PM) (measured 90 min after shift to the permissive temperature) was completely inhibited by high expression of CFP-Sec16 (Figure 4B). In cells expressing these high levels of GFP-Sec16, the Golgi apparatus remains intact, indicating a block in ER export rather than simply a disrupted Golgi apparatus (Figure 4C, D).

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