Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network

Abstract

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.

Figure 1.

Reconstitution of the EE/RE-TGN transport by using a modified STxB. (A) Transport was carried out in the absence or presence of 0.6, 1.2, 1.8, 2.4, and 3.0 mg/ml rat liver cytosol as indicated. The relative level of transport was shown, with the highest transport level being defined arbitrarily as 100%. (B-D) Standard transport mixtures containing perforated cells, rat liver cytosol, and ATP regeneration system were preincubated for 1 h on ice with BFA (B), nocodazol (B), GTPγS (C), or various antibodies (D) as indicated. They were then shifted to 37°C and incubated for a further 90 min. Controls were the standard transport setups without any additional reagents or antibodies, and the levels of transport were arbitrarily defined as 100%. The respective concentrations (micrograms per milliliter) of the added reagents were shown in brackets (B and D). H, heat inactivated.

Figure 4.

STxB transport to the TGN was inhibited in HeLa cells when GS15 expression is knocked down by its siRNA. HeLa cells grown on 24-well plates were transfected with GS15 or control siRNA for 48 h followed by Western blot analysis with antibodies against GS15, Bet1, GS27, or β-tubulin (A) or analysis of the levels of ³⁵S-labeled STxB (assessed by in vivo transport) and β-tubulin (assessed by Western blot) after the in vivo STxB transport assay (B).

Figure 5.

Two SNARE complexes are involved in the EE/RE-TGN transport. (A) Inhibition by syntaxin 5 and syntaxin 16 antibodies was additive. (B) Inhibition by syntaxin 5 and GS15 antibodies was not additive, whereas the inhibition by syntaxin 16 and GS15 antibodies was. (A and B) Transport was carried out in the presence of buffers (controls), antibodies, or combination of two different antibodies as indicated. (C) Inhibition by GS15 siRNA in vivo and syntaxin 16 antibody in vitro was additive. HeLa cells transfected with GS15 or control siRNA were processed to make semi-intact cells and used for in vitro transport in the presence or absence of syntaxin 16 antibodies as indicated. (A-C) Extent of transport relative to each control (set as 100%) was calculated from two to three separate experiments. Error bars represented standard deviations. The respective concentrations (micrograms per milliliter) of the added reagents were shown in brackets.

Figure 6.

Antibodies against syntaxin 5 and syntaxin 16 exhibited similar kinetics of inhibition. Standard transport reactions at 37°C were allowed to proceed for 0, 5, 10, 15, 20, 30, 45, and 90 min as indicated and paused on ice. Either 67 μg/ml syntaxin 5 or 20 μg/ml syntaxin 16 antibodies were added at these time points, and the mixture was left on ice for 50 min. Transport reactions were resumed at 37°C and allowed to proceed until 90 min in total. (A) Representative phosphorimaging data. (B) Graph plotted from duplicates. Maximum inhibition values were set as 100%. This result suggests that on its way from endosomes to the TGN, STxB passes syntaxin 5- and syntaxin 16-sensitive stages simultaneously.

Figure 7.

Syntaxin 16-mediated (syntaxin 5-independent) and syntaxin 5-mediated (syntaxin 16-independent) routes have similar time courses of transport. Transport reactions with either 67 μg/ml syntaxin 5 or 20 μg/ml syntaxin 16 antibodies were allowed to proceed for 10, 15, 20, 30, 40, 50, 60, and 70 min at 37°C and stopped on ice. Controls (cont.) were transport reactions that proceeded for 70 min in the absence of any antibody. Transport relative to control (set as 100%) at each time point was calculated. (A) Representative phosphorimaging data. (B) Graph plotted from duplicates. Curves fitting each set of data are presented.

Figure 2.

Syntaxin 5, GS28, Ykt6, and GS15 are involved in the EE/RE-TGN transport. (A-C) Syntaxin 5, GS15, and Ykt6 polyclonal antibodies inhibited the EE/RE-TGN transport. (D) GS28, but not GS27 mAb inhibited the in vitro transport. As indicated, transport reactions were carried out in the presence of buffer (controls), antigens, GST, antibodies, or heat-inactivated antibodies. Where both antibodies and antigens (or GST) were present, antibodies were incubated with antigens (or GST) on ice for 1 h before being added to the standard reactions. Extent of transport relative to each control (defined as 100%) was an average from two to four separate experiments. Error bars represented standard deviations. The respective concentrations (micrograms per milliliter) of the added reagents were shown in brackets. H, heat inactivated.

Figure 3.

Fab fragments derived from syntaxin 5 or GS15 antibodies inhibited the EE/RE-TGN transport. Fab fragments were prepared from syntaxin 5 or GS15 antibodies. Transport was carried out in the presence of buffer (controls), Fab fragments, antigens, or heat-inactivated Fab fragments. Where both Fab fragments and antigens were present, Fab fragments were incubated with antigens on ice for 1 h before being added to the transport reactions. The extent of transport relative to each control (defined as 100%) was calculated from two to three separate experiments. (A) Transport in the presence of the Fab fragments and/or antigens of syntaxin 5 antibodies. (B) Transport in the presence of the Fab fragments and/or antigens of GS15 antibodies. Error bars represented standard deviations. The respective concentrations (micrograms per milliliter) of the added reagents were shown in brackets. H, heat inactivated.

Figure 8.

Some GS15 and Ykt6 were redistributed to endosomes in SNX3-overexpressing cells. A431 cells were transfected with myc-SNX3 and double labeled with antibodies against GS15 (B), Ykt6 (E), syntaxin 5 (G), or GS27 (J) and anti-Myc antibody in SNX3-overexpressing cells (A, D, F, and I). Bar, 10 μM.