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. 2002 Oct;13(10):3493-507.
doi: 10.1091/mbc.e02-01-0004.

GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus

Yue Xu  1 , Sally Martin, David E James, Wanjin Hong
Affiliations

GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus

Yue Xu et al. Mol Biol Cell. 2002 Oct.

Abstract

The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. In our present study, it is revealed that unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15 degrees C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis- and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus.

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Figures

Figure 1
Figure 1
GS15 does not recycle back to the IC. NRK cells cultured at 37°C (A: a–c; B: g–i), preincubated at 15°C for 3 h (A: d–f; B: j–l), or pretreated with brefeldin A at 10 μg/ml for 60 min (B: m–o) were fixed and processed for double-labeling to reveal the distribution of GS15 and the KDEL receptor (KDEL-R) (A) or GS15 and Bet1 (B). As shown, 15°C incubation resulted in a shift of KDEL-R and Bet1 from the Golgi to peripheral IC, while having no effects on GS15 distribution in the compact Golgi. Brefeldin A redistributed GS15 to diffuse ER-like structure, while Bet1 was redistributed into more peripheral dotted structures. Bar, 10 μM.
Figure 2
Figure 2
GS15 behaves the same as Golgi mannosidase II (ManII). NRK cells cultured at 37°C (panels a–c) or preincubated at 15°C for 3 h (panels d–f) were fixed and processed for double-labeling to reveal the distribution of GS15 and ManII. As shown, both GS15 and ManII are distributed in compact Golgi structure at both temperatures. Bar, 10 μM.
Figure 3
Figure 3
GS15 is enriched in the medial cisternae and adjacent vesicular-tubular structures. Cryosections derived from indicated cells or tissues were processed for immunogold labeling at the electron microscopy level. As shown, GS15 is detected mainly on medial cisternae and its associated vesicular-tubular structures in CHO cells, 3T3-L1 adipocytes, and spermatid. Size bar, 100 nm.
Figure 4
Figure 4
GS15 is detected on vesicular-tubular profiles marked by COPI coat in the medial cisternae of the Golgi. Cryosections derived from pancreas was double-labeled with antibodies against GS15 (revealed by 10 nm gold particles) and mAb against β-COP (revealed by 5-nm gold particles). As shown, GS15 and β-COP could be seen in the vicinity of the same vesicular-tubular structures (indicated by arrows). Size bar, 100 nm.
Figure 5
Figure 5
GS15 is present at high concentrations on isolated Golgi cisternae. Isolated Golgi apparatus were double-labeled with antibodies against GS15 (revealed by 10-nm gold particles) and mAb against the KDEL receptor (p23) (revealed by 5-nm gold particles). The whole-mount Golgi cisternae were examined by EM. Size bar, 200 nm.
Figure 6
Figure 6
Reciprocal coimmunoprecipitation of GS15, syntaxin 5, GS28 and Ykt6. A: Golgi detergent extracts were immunoprecipitated with antibodies against GS15 (lane 1) or control antibodies (lane 2). The immunoprecipitates of anti-GS15 (lane 1) or control antibodies (lane 2) and 10% of the starting material (lanes 3) were resolved by SDS-page and processed for immunoblot to detect the respective proteins as indicated on the left. As shown, syntaxin 5, GS28 and Ykt6, but not Bet1, Sec22b or syntaxin 6 were coimmunoprecipitated by anti-GS15 antibodies. B: Golgi detergent extracts were immunoprecipitated with antibodies against GS28 (lane 2), syntaxin 5 (lane 3), Ykt6 (lane 4), or control antibodies (lane 1). The immunoprecipitates, along with 10% starting material (S) were resolved by SDS-page and analyzed by immunoblot to detect the respective proteins as indicated on the left. C. Golgi detergent extracts were immunoprecipitated with antibodies against syntaxin 5 (lanes 1 and 3) or control antibodies (lanes 2 and 4). The immunoprecipitates (lanes 1 and 2) and 10% supernatants (lanes 3 and 4) as indicated were resolved by SDS-page and processed for immunoblot to detect the indicated proteins.
Figure 7
Figure 7
Coimmunoprecipitation of components of COPI coat by anti-GS15 antibodies. A: Golgi detergent extracts were immunoprecipitated with control antibodies (lane 1) or anti-GS15 antibodies (lane 2). The immunoprecipitates were resolved by SDS-page. After staining with Coomassie-blue, the proteins bands immunoprecipitated by anti-GS15 were excised for amino acid sequence identification. The amino acid sequences of the indicated proteins were shown on the right. B. Golgi detergent extracts were immunoprecipitated with antibodies against GS15 (lanes 1), anti-Bet1 (lane 3), or control antibodies (lanes 2 and 4). The immunoprecipitates were resolved by SDS-page and processed for immunoblotting with antibodies against β-COP.
Figure 8
Figure 8
Expression of GS15 mutant without TM (GS15ΔTM) disrupts normal Golgi distribution of syntaxin 5, GS28, and Man II, while the Golgi distribution of Vit1-rp2/Vit1a, syntaxin 6 and Golgi matrix GM130 and p115 were less affected. NRK cells were transiently transfected with a construct for expressing GS15ΔTM. Transfected cells were fixed and processed for indirect immunofluorescence to detect the expressed mutant GS15 (left panels) and indicated Golgi proteins in central panels. The merged images are presented. Bar, 10 μM.
Figure 9
Figure 9
Expression of GS15(Q49A) mutant affects normal Golgi distribution of syntaxin 5, GS28, and Man II. NRK cells were transiently transfected with a construct for expressing GS15(Q49A). Transfected cells were fixed and processed for indirect immunofluorescence to detect the expressed mutant GS15 (left panels) and indicated Golgi proteins in center panels. The merged images are presented in the right panels. Bar, 10 μM.
Figure 10
Figure 10
An essential role of GS15 in the Golgi apparatus. Hela cells were transfected with GS15-specific siRNA and then analyzed by immunofluorescence microscopy to detect GS15 and other indicated markers of the Golgi apparatus. (A) cis/medial proteins GS28, syntaxin 5, and Ykt6 were redistributed into small dotty and diffuse labeling in cells whose GS15 levels were significantly reduced by siRNA. (B) Even trans-Golgi/TGN β1,4-galactosyltransferase (GT), TGN syntaxin 6, and Golgi matrix GM130 were noticeably affected by GS15 knockdown. Bars: 10 μM.
Figure 10
Figure 10
An essential role of GS15 in the Golgi apparatus. Hela cells were transfected with GS15-specific siRNA and then analyzed by immunofluorescence microscopy to detect GS15 and other indicated markers of the Golgi apparatus. (A) cis/medial proteins GS28, syntaxin 5, and Ykt6 were redistributed into small dotty and diffuse labeling in cells whose GS15 levels were significantly reduced by siRNA. (B) Even trans-Golgi/TGN β1,4-galactosyltransferase (GT), TGN syntaxin 6, and Golgi matrix GM130 were noticeably affected by GS15 knockdown. Bars: 10 μM.
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