Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events - PubMed (original) (raw)
Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events
Paul R Pryor et al. EMBO Rep. 2004 Jun.
Abstract
Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.
Figures
Figure 1
SNAREs in the late endocytic pathway. (A) Immunoblots of SDS–PAGEseparated SNAREs from late endosome- and lysosome-enriched membrane fractions from the rat liver. 20 μg protein/track. (B) Quantification of gold particles per micrometre limiting membrane of late endosomes and lysosomes after immunogold labelling with anti-SNARE antibodies. Data for Syn7 and V8 from Mullock et al (2000) and the same EM blocks were used to localize the other SNAREs. (C) Immunoelectron micrograph showing labelling of VAMP7 on lysosomes. Gold particles, 10 nm; scale bar, 0.5 μm. Syn7, syntaxin 7; Vti, Vti1b; Syn8, syntaxin 8; V8, VAMP8; V7, VAMP7; LE, late endosome; Ly, lysosome.
Figure 2
Cell-free fusion assays. (A) Time courses of content mixing assays for late endosome–lysosome fusion and homotypic late endosome fusion using membrane fractions from the rat liver. (B) Inhibition of fusion by affinity-purified antibodies against SNAREs. Standard fusion assays were run for 10 min at 37°C in the presence of 100 μg/ml affinity-purified antisNARE antibodies. Representative experiments (means±range of pairs) are shown in which the same affinity-purified antibody preparations were used in each of the two assays on the same day with the same late endosome preparations containing avidin–ASF.
Figure 3
Combinatorial SNARE complexes. (A) Schematic representation of combinatorial SNARE interactions late in the endocytic pathway with the same t-(Q-)SNARE complex interacting with different v-(R-)SNAREs (V8 and V7) to allow, respectively, homotypic fusion of late endosomes or heterotypic fusion with lysosomes. Although not shown, individual SNAREs are present, and may be functional, on either membrane. (B) Immunoprecipitation of SNAREs from solubilized liver membrane fractions followed by SDS–PAGE and immunoblotting.
Figure 4
Effect of overexpressing the longin domain of VAMP7 on traffic through the endocytic pathway of NRK cells. (A) Distribution of Texas Red–dextran after uptake for 4 h followed by 20 h chase. (B) Immunofluorescence localization of lgp120 in the same cell as in (A). (C) Colocalization (yellow) of Texas red–dextran (A, red) and lgp120 (B, green). (D) Expression of GFP–V7 longin domain (24 h) in a cell subsequently pulse-chased (4 h pulse, 20 h chase) with biotinylated dextran (E). (F) Immunofluorescence localization of EEA1 (red) in the same cell shown in (E) containing biotinylated dextran (blue). (G–L) Distribution of Texas Red–dextran (G), lgp120 (H) and biotinylated dextran (J) in a cell preloaded with Texas Red–dextran (4 h pulse, 20 h chase), transfected with a plasmid encoding GFP–longin domain of VAMP7 (L), and after 24 h, incubated with biotinylated dextran (4 h uptake, 20 h chase). (I) Colocalization (yellow) of Texas Red–dextran (G, red) and lgp120 (H, green). (K) Colocalization (magenta) between biotinylated dextran (J, blue) and Texas Red–dextran (G, red). Scale bar, 10 μm.
Figure 5
Inhibition of delivery to lysosomes by the VAMP7 longin domain. NRK cells were preloaded with BSA–5 nm gold (4 h pulse, 20 h chase). They were then transiently transfected with a plasmid encoding GFP–longin domain of VAMP7 (V7 LD), or GFP–VAMP8 (V8), and after 24 h allowed to take up BSA–15 nm gold (4 h pulse, 20 h chase). (A) After EM, the percentage of organelles containing BSA–15 nm gold that were electron dense/contained (closed bars), or electron lucent/did not contain (open bars), BSA–5 nm gold measured in cells expressing V7 LD or V8, or surrounding cells not expressing GFP (control). The data shown are mean±s.e.m. from single sections of six control and six V7 LD-transfected cells from two separate experiments and four V8-transfected cells from a different experiment (surrounding nontransfected cells had a distribution of gold particles not significantly different from the control shown). (B–D) Representative images of main organelles containing 15 nm gold in transfected and control cells (large arrows, 15 nm gold; small arrows, 5 nm gold). (E) Cell-free late endosome–lysosome fusion after addition of bacterially expressed his6-tagged V7 longin domain or GST as a control (*P<0.03 versus GST control). Data are means±s.e.m., _n_=4.
References
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