Research Article

The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins

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Science  31 Oct 2014:
Vol. 346, Issue 6209, 1256898
DOI: 10.1126/science.1256898

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Structured Abstract

Introduction

The eukaryotic cell contains membrane-bound organelles with distinct functionality and composition. Preservation of organelle identity depends on the highly selective transfer of proteins and lipids between compartments. Central to this are transport carriers called vesicles. Mechanisms are required not only for the selective incorporation of specific cargos into vesicles as they bud off a donor organelle, but also for the correct delivery to an acceptor organelle. SNARE proteins on the vesicle and destination organelle drive membrane fusion after arrival and have been implicated in contributing to specificity in choice of organelle. However, upstream of the fusion step, a process called tethering is thought to initially attach the vesicle to the destination organelle and then bring it close to allow the SNARE proteins on opposite membranes to interact. The importance of tethering in conferring specificity to membrane traffic is currently unclear.

Embedded Image

Golgins are sufficient to capture specific classes of vesicle. We hypothesized that if the golgins tether vesicles destined for the Golgi, then their relocation to mitochondria should result in ectopic capture of specific classes of vesicle. Immunofluorescence demonstrates that the presence of a single golgin on mitochondria results in the ectopic capture of a specific cargo. Electron microscopy reveals that vesicle s accumulate around these mitochondria.

Rationale

To study the contribution of tethering to specificity in membrane trafficking, we focused on the Golgi apparatus. The Golgi complex is a multicompartment organelle at the intersection of secretory and endocytic trafficking pathways and so receives vesicles from a range of destinations. A family of well-conserved large coiled-coil proteins on the Golgi, the golgins, have been suggested to function as vesicle tethers at the Golgi. However, mild phenotypes of golgin mutants have presented a challenge for elucidating their in vivo roles. We thus used a relocation strategy to test for their sufficiency rather than necessity in vesicle tethering. Ten mammalian golgins that are conserved outside of vertebrates and found on different regions of the Golgi were ectopically expressed at the mitochondria through attachment to a mitochondrial transmembrane domain in place of their C-terminal Golgi targeting domain. We then used the distribution of cargo-laden vesicles originating from different locations as a readout for the golgins’ tethering activity.

Results

We demonstrate that subsets of golgins are capable of redirecting particular endogenous or exogenous cargo destined for the Golgi to an ectopic site, the mitochondria. Specifically, golgin-97, golgin-245, and GCC88 were able to capture endosome-to- Golgi cargos; GM130 and GMAP210 were able to capture endoplasmic reticulum (ER)–to-Golgi cargos; and golgin-84, TMF, and GMAP210 were able to capture Golgi resident proteins. Furthermore, electron microscopy yielded ultrastructural evidence for the accumulation of vesicular membranes around mitochondria decorated with specific golgins. These data suggest that not only do the golgins capture vesicles, but they also exhibit specificity toward vesicles of different origins—from the endosomes, from the ER, or from within the Golgi itself.

Conclusion

We have been able to demonstrate that relocation of specific golgins is sufficient to reroute specific classes of transport vesicles to an ectopic site. Thus, most golgins are sufficient to nucleate a specific tethering process, and hence they are likely to make a major contribution to the specificity of vesicle traffic arriving at the Golgi. In addition, this relocation system may be a useful tool for isolating specific transport vesicles that are normally short-lived, hence providing a route to further understanding of specificity in membrane traffic.

You've got to pick a Golgi tether or two

The inside of the cell contains a large variety of different membrane transport vesicles, each of which needs to find and fuse with its correct target destination. The detailed mechanism specifying which vesicle can fuse with which target membrane has been the subject of an enormous amount of research. An additional layer of specificity in intracellular membrane trafficking across the Golgi complex is thought to involve particular membrane “tethers.” However, the importance of these tethers has been unclear. Wong and Munro used a clever trick to reveal how specific tethers can indeed ensure correct vesicle destination. Tether proteins experimentally expressed on mitochondria hijacked different transport vesicles and diverted them from their normal destination to the mitochondria.

Science, this issue 10.1126/science.1256898

Abstract

The Golgi apparatus is a multicompartment central sorting station at the intersection of secretory and endocytic vesicular traffic. The mechanisms that permit cargo-loaded transport vesicles from different origins to selectively access different Golgi compartments are incompletely understood. We developed a rerouting and capture assay to investigate systematically the vesicle-tethering activities of 10 widely conserved golgin coiled-coil proteins. We find that subsets of golgins with distinct localizations on the Golgi surface have capture activities toward vesicles of different origins. These findings demonstrate that golgins act as tethers in vivo, and hence the specificity we find to be encoded in this tethering is likely to make a major contribution to the organization of membrane traffic at the Golgi apparatus.

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