Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack - PubMed (original) (raw)

Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack

Allen Volchuk et al. Mol Biol Cell. 2004 Apr.

Abstract

Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.

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Figures

Figure 1.

Figure 1.

Localization of GS15 in NRK cells by immunofluorescence microscopy. Rat NRK cells were fixed and immunostained for GS15 and Mannosidase II (top panels) or GS15 and TGN38 (bottom panels) and analyzed by laser confocal microscopy as described in MATERIALS AND METHODS. Areas of colocalization of the two proteins in the merged images appear yellow.

Figure 2.

Figure 2.

Localization of Ykt6 in Hela cells by immunofluorescence microscopy. (A) Human Hela cells were fixed and immunostained with a goat anti-Ykt6 antibody and a rabbit anti-GOS28 antibody as described in MATERIALS AND METHODS. (B) Human Hela cells were stained for Ykt6 before (Control) and after treatment with brefeldin A (10 μg/ml for 30 min; BFA). (C) Hela cell total lysate (60 μg) was resolved by SDS-PAGE and immunoblotted with a goat anti-Ykt6 antibody.

Figure 7.

Figure 7.

Relative molar amounts of Golgi SNAREs in cells. Rat NRK cells were uniformly labeled with 35[S]methionine as described in MATERIALS AND METHODS. Individual SNARE proteins were immunoprecipitated and resolved by SDS-PAGE. The amount of each 35S-labeled SNARE in arbitrary units was determined using a Molecular Dynamics PhosphorImager. The absolute abundance (pmol/mg cell protein) of the individual SNAREs were calculated as stated in MATERIALS AND METHODS and are as follows: rBet1, 317 ± 9; GS15, 308 ± 61; ERS24, 295 ± 39; syntaxin 5 (34 kDa), 133 ± 28; membrin, 101 ± 2; GOS28, 63 ± 10. Values were obtained from three independent experiments and shown are the mean ± SD normalized to syntaxin 5. SYNT5, syntaxin 5 (34-kDa Golgi form).

Figure 6.

Figure 6.

Distribution of SNAREs within the IC, Golgi stack, and TGN. The distribution of the indicated SNAREs within the IC, Golgi stack (cisterna 1 (cis) to cisterna 5 (trans)), and TGN, were obtained as outlined in MATERIALS AND METHODS. Shown is the linear density of labeling (gold particles/μm membrane) within these compartments. For each SNARE its distribution was normalized to the value in the compartment having the highest labeling density. (A) Data for the subunits of the _cis_-SNAREpin, v-rBet1, and light chains membrin and ERS24. t-LC, t-SNARE light chain; v, v-SNARE. (B) Data for the subunits of the _trans_-SNAREpin, v-GS15, and t-SNARE heavy chain syntaxin 5 and t-light chain GOS28 (t-HC, t-SNARE heavy chain). Error bars, SEM

Figure 3.

Figure 3.

Ultrathin cryosections of NRK cells showing immunogold labeling of GS15 and GOS28. GS15 is preferentially localized over the medial and trans cisternae of the Golgi stack, whereas GOS28 is distributed across the stack. The quantitative evaluation of the labeling is shown in Tables 1 and 2. Bar, 100 nm.

Figure 4.

Figure 4.

Ultrathin cryosections of NRK cells showing immunogold labeling of membrin, ERS24, and syntaxin 5. Membrin and ERS24 distribute mostly to the cis side of the Golgi, including elements of the intermediate compartment, whereas syntaxin 5 is present throughout the entire Golgi stack. The quantitative evaluation of the labeling is shown in Tables 1 and 2. Bar, 100 nm.

Figure 5.

Figure 5.

Assessment of the _cis_-trans polarity of the Golgi stack. Double labeling of GM130 and ERS24 (A), GM130 and membrin (B), GM130 and GS15 (C). GM130, a _cis_-Golgi marker, colocalizes with ERS24 and membrin but not with GS15, a SNARE localized to the _medial/trans_-Golgi. GM130 was visualized with small (10 nm) gold particles (arrowhead), whereas ERS24, membrin, and GS15 were revealed by large (15 nm) gold particles (arrow). Bar, 100 nm.

Figure 8.

Figure 8.

Concentration of SNAREs in peri-Golgi vesicles relative to the common Golgi t-SNARE heavy chain, syntaxin 5. (A) Shown for each SNARE is the ratio of the linear density (gold particles/μm) within lateral peri-Golgi vesicles (from Table 2) relative to the linear density in the Golgi stack (from Table 2). SYNT5, syntaxin 5 (34-kDa Golgi form). (B) Shown is the calculated mole ratio of the various SNAREs to syntaxin 5 in peri-Golgi vesicles. The values were obtained by multiplying the values in Figure 7 (mole ratios of the SNAREs in cells) by the values in panel A (linear density of gold particles in peri-Golgi vesicles/Golgi stack), and renormalizing to syntaxin 5.

Figure 9.

Figure 9.

Requirement for SNAREs in cell-free reconstituted Golgi transport. Transport-coupled glycosylation of VSV-G protein was measured. Purified Golgi membranes from VSV-infected 15B cells (donor) and wild-type CHO cells (acceptor), were incubated with cytosol and ATP as described in MATERIALS AND METHODS. The assay was performed with increasing concentrations of the indicated Fab fragments or (as controls) nonspecific rabbit Fab or a Fab directed against Vti1b, as indicated. Triplicate samples were used for each condition generating the SDs shown. SYNT5, syntaxin 5.

Figure 10.

Figure 10.

Countercurrent distribution of SNAREpins and its implication for vesicle transport patterns in the Golgi stack. Distinct cis_- and trans_-Golgi SNAREpins are needed for the operation of the Golgi stack. They distribute in countercurrent concentration gradients across the stack. Two types of COPI vesicles bud at every level of the Golgi stack (Orci et al., 1997). One contains retrograde-directed cargo and the cis_-Golgi v- and t-SNAREs (v_cis and t_cis). The other contains anterograde-directed cargo and the trans_-Golgi v- and t-SNAREs (v_trans and t_trans). Both vesicles fuse homotypically with cognate SNAREs. They are restrained from dissociating by a carpet of tethers (Orci et al., 1998; Sonnichsen et al., 1998; Seemann et al., 2000) and therefore are restricted to fusion with adjacent cisternae on either side. With these conditions, at each level of the stack a vesicle will have a higher probability of fusing to the neighboring cisternae that contains the higher—rather than the lower—concentration of its cognate (homotypic) SNAREs. The opposing _cis_-trans distribution of the two cognate SNARE pairs would mean that the “retrograde” vesicles carrying _cis_-Golgi SNAREs should have a systematically higher probability of fusing up their gradient toward the cis face, whereas the “anterograde” vesicles carrying _trans_-Golgi SNAREs would, to the contrary, fuse preferentially up their gradient toward the trans face. In other words, the two types of vesicles would engage in systematically biased random walks toward the opposite ends of the stack, as individual vesicles essentially chromatograph up their gradients. This mechanism would naturally create a net flow of the cargo contained in the “anterograde” vesicles across the stack in the _cis_-to-trans direction, and a net flow of the cargo contained in the “retrograde” vesicles in the opposite _trans_-to-cis direction (this model is a refinement of the “percolating vesicle” model which envisioned an unbiased random walk; Pelham and Rothman, 2000). The countercurrent of cargo could of course be fine-tuned by additional layers of specificity involving the distributions of regulatory proteins like rabs and tethers. This model for vesicular transport in the stack does not speak to the cisternal progression/maturation process that likely occurs concurrently (Pelham and Rothman, 2000).

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References

    1. Balch, W.E., Dunphy, D.G., Braell, W.A., and Rothman, J.E. (1984a). Reconstitution of the transport of protein between successive compartments of the Golgi measured by the incorporation of N-acetyl glucosamine. Cell 39, 405-416. - PubMed
    1. Balch, W.E., Glick, B.S., and Rothman, J.E. (1984b). Sequential intermediates in the pathway of intercompartmental transport in a cell-free system. Cell 39, 535-536. - PubMed
    1. Banfield, D.K., Lewis, M.J., and Pelham, H.R. (1995). A SNARE-like protein required for traffic through the Golgi complex. Nature 375, 806-809. - PubMed
    1. Barbero, P., Bittova, L., and Pfeffer, S.R. (2002). Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells. J. Cell Biol. 156, 511-518. - PMC - PubMed
    1. Blagitko, N., Schulz, U., Schinzel, A.A., Ropers, H.H., and Kalscheuer, V.M. (1999). γ2-COP, a novel imprinted gene on chromosome 7q32, defines a new imprinting cluster in the human genome. Human Mol. Genet. 8, 2387-2396. - PubMed

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