Countercurrent Distribution of Two Distinct SNARE Complexes Mediating Transport within the Golgi Stack

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. INTRODUCTIONIntracellular membrane fusion results from the assembly of a target membrane t-SNARE, composed of one heavy chain (syntaxin) and two distinct light chains, with a cognate vesicle membrane v-SNARE (Sollner et al., 1993;Weber et al., 1998;Hu et al., 2003). The resulting SNAREpin, held together by a bundle of four ␣-helices (Sutton et al., 1998), forcefully perturbs the apposing lipid bilayers, thereby triggering fusion (McNew et al., 2000b). Cognate SNARE pairing between bilayers is the immediate determinant of the specificity of membrane fusion McNew et al., 2000a;Parlati et al., 2000Parlati et al., , 2002Paumet et al., 2001). The v-SNARE is functionally distinguished from the other subunits of the SNAREpin because fusion can only occur when the v-SNARE resides in the opposite bilayer from the remaining subunits, which comprise the t-SNARE .In particular, fusion of vesicles derived from the ER with the Golgi requires the pairing of the v-SNARE Bet1 (in yeast; rBet1 in animals) with the t-SNARE comprised of the syntaxin heavy chain Sed5 (syntaxin 5) and light chains Bos1 (membrin) and Sec22 (ERS24, also named Sec22b; , abbreviated t-Sed5/Bos1, Sec22. The genes encoding these four "ER-Golgi" SNAREs are required for ER-toGolgi transport in yeast in vivo (Newman and Ferro-Novick, 1987;Newman et al., 1990;Shim et al., 1991;Hardwick and Pelham, 1992;Cao and Barlowe, 2000). Liposomes bearing the v-SNARE Bet1 only fuse with liposomes bearing the cognate t-SNARE Sed5/Bos1, Sec22 (Parlati et al., , 2002Paumet et al., 2001).The intracellular distribution of these SNAREs determined by immunoelectron microscopy fits well with their roles in ER-to-Golgi transport in animal cells (Paek et al., 1997;Hay et al., 1998;Orci et al., 2000;Martinez-Menarguez et al., 2001). The "short" Golgi-localized form of the t-SNARE heavy chain syntaxin 5 (Hui et al., 1997), which lacks the ER-retrieval signal encoded in the additional exon of the "long" form, is located in every cisternae across the Golgi stack i...

Differential use of endoplasmic reticulum membrane for phagocytosis in J774 macrophages

“…Anti-ERS24 Fab fragments have been shown to efficiently block ERS24 function in intra-Golgi transport (18). Introduction of anti-ERS24 Fab fragments into J774 cells also inhibited phagocytosis of large beads in a dose-dependent manner (Fig.…”

“…ERS24 is required for ER-Golgi transport (17) and intra-Golgi transport (18) where it functions as a subunit of a Golgi-localized target-SNARE (12). Therefore, the observed requirement of ERS24 for phagocytosis might be secondary to the inhibition of the secretory pathway.…”

“…ERS24-mediated ER-plasma membrane fusion in support of phagocytosis would require the recruitment of ERS24-containing ER membranes to sites of internalization. In resting cells, ERS24 is localized in the ER and Golgi (18). To ascertain which ERS24-containing compartment(s) is recruited for phagosome formation, we studied the localization of other SNARE proteins during phagocytosis of 3.0-m beads.…”

Dynamic transport of SNARE proteins in the Golgi apparatus

“…The determination of the cis-trans distribution of endogenous Golgi SNAREs is described in ref. 4. For endogenous markers, cis-Golgi SNAREs exhibit a high rim͞center ratio and trans-Golgi SNAREs a lower rim͞center ratio.…”

“…It controls the fusion of endoplasmic reticulum (ER)-derived vesicles with the cis-Golgi, and it is generally believed that Bet1 is the v-SNARE subunit in this complex (1), although some have proposed that ERS24 might be the v-SNARE (2,3). At least one other SNARE complex (GS15-GOS28-syntaxin5-Ykt6) is present in the Golgi apparatus, but this complex is more concentrated on the trans face and might control intra-Golgi transport (either anterograde or retrograde) (4), or possibly transport between the Golgi apparatus and endosomes (5).…”