Coiled-coil interactions are required for post-Golgi R-SNARE trafficking - PubMed (original) (raw)

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Coiled-coil interactions are required for post-Golgi R-SNARE trafficking

David E Gordon et al. EMBO Rep. 2009 Aug.

Abstract

The sorting of post-Golgi R-SNAREs (vesicle-associated membrane protein (VAMP)1, 2, 3, 4, 7 and 8) is still poorly understood. To address this, we developed a system to investigate their localization, trafficking and cell-surface levels. Here, we show that the distribution and internalization of VAMPs 3 and 8 are determined solely through a new conserved mechanism that uses coiled-coil interactions, and that VAMP4 does not require these interactions for its trafficking. We propose that VAMPs 3 and 8 are trafficked while in a complex with Q-SNAREs. We also show that the dileucine motif of VAMP4 is required for both its internalization and retrieval to the trans-Golgi network. However, when the dileucine motif is mutated, the construct can still be internalized potentially through coiled-coil interactions with Q-SNAREs.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Schematic of VAMP-HA constructs. (A) VAMP-HA constructs were generated by two-step PCR (TM, transmembrane domain; HA-serine linker and HA tag). Numbers indicate amino acids positions. The VAMP4 dileucine motif (underlined) was mutated to AA (V4AA). (B) Alignment of the coiled-coil domains of VAMPs 3–8. Conserved hydrophobic residues are highlighted in grey; these residues were mutated to proline to generate the coiled-coil mutants (P). Residues marked with an asterisk were mutated to AA in VAMP3 (ADIA). HA, haemagglutinin; VAMP, vesicle-associated membrane protein.

Figure 2

Figure 2

VAMP4 can be internalized through its dileucine motif or its coiled-coil domain. (A) Expression levels of VAMP4-HA constructs were determined by Western blotting. Extracts from HeLa cells stably transduced with empty vector (mock), wild-type (WT) and mutant constructs (AA, P and AA+P) were probed with HA, VAMP4 (V4) and γ antibodies (AP-1, γ was used as a loading control). (B) To determine the steady state localization of the VAMP4-HA constructs, immunofluorescence microscopy was performed on fixed cells stained with HA antibodies. For HA-uptake experiments, cells were incubated in the presence of HA antibodies for 4 h at 37°C before being fixed. Scale bar, 10 μm. (C) To measure SNARE complex formation immunoprecipitations (anti-HA) were performed on detergent extracts generated from wild-type and mutant VAMP4-HA cells. Blots were probed with HA, syntaxin (STX) 6, 7 and 8 antibodies. (D) Cell surface levels of VAMP4-HA constructs were measured by flow cytometry. The mean fluorescence for each cell population was calculated. The data shown are the average of three experiments, ±s.e. AA, dileucine mutant; HA, haemagglutinin; IP, immunoprecipitation; P, proline mutant coiled-coil; VAMP, vesicle-associated membrane protein; WB, Western blotting.

Figure 3

Figure 3

VAMPs 3 and 8 are trafficked through their coiled-coil domains. (A) Expression levels of VAMP-HA constructs were determined by Western blotting. Extracts generated from HeLa cells stably transduced with empty vector (mock), wild-type and proline mutant coiled-coil (P) constructs, were probed with HA, VAMPs 3, 7, 8 and γ antibodies (AP-1, γ was used as a loading control). (B) To measure SNARE complex formation immunoprecipitations (IPs; anti-HA) were performed on detergent extracts generated from wild-type or mutant VAMP-HA cells. Blots were probed with HA, syntaxin (STX) 6, 7 and 8 antibodies. (C) To determine the steady state localization of the VAMP-HA constructs, immunofluorescence microscopy was performed on fixed cells stained with HA antibodies. For HA-uptake experiments, cells were incubated in the presence of HA antibodies for 4 h at 37 °C before being fixed. Scale bar, 10 μm. (D) Cells surface levels of VAMP-HA constructs were measured by flow cytometry. The mean fluorescence for each cell population was calculated. The data shown are the average of three experiments, ±s.e. HA, haemagglutinin; P, proline mutant coiled-coil; VAMP, vesicle-associated membrane protein; WB, Western blotting.

Figure 4

Figure 4

Mutation of VDIM to ADIA in the coiled-coil domain of VAMP3 causes defects in SNARE complex formation and trafficking. (A) Expression levels of VAMP3-HA constructs were determined by Western blotting. Extracts generated from HeLa cells stably transduced with empty vector (mock), wild-type (WT), coiled-coil mutant (P) and 26VDIM29 to 26ADIA29 mutant (ADIA) constructs were probed with HA, VAMP3 and γ antibodies (AP-1, γ was used as a loading control). (B) To measure SNARE complex formation immunoprecipitations (IP; anti-HA) were performed on detergent extracts generated from HeLa cells stably expressing wild-type and mutant VAMP3-HA constructs. Blots were probed with HA, syntaxin (STX) 6, 7 and 8 antibodies. (C) To determine the steady state localization of the VAMP3-HA constructs, immunofluorescence microscopy was performed on fixed cells stained with HA antibodies. For HA-uptake experiments, cells were incubated in the presence of HA antibodies for 4 h at 37 °C before being fixed. Scale bar, 10 μm. (D) Cells surface levels of VAMP3-HA constructs were measured by flow cytometry. The mean fluorescence for each cell population was calculated. The data shown are the average of three experiments, ±s.e. HA, haemagglutinin; P, proline mutant coiled-coil; VAMP, vesicle-associated membrane protein; WB, Western blotting.

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