Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.     

Making COPII coats

Newly synthesized proteins en route to the Golgi apparatus are exported from the endoplasmic reticulum by COPII coated vesicles. Fath et al. (2007) now reveal the structure of a large portion of the yeast Sec13/31 complex, which comprises the coat framework of COPII-coated vesicles. Their findings suggest a mechanism by which the COPII cage assembles and accommodates cargo of different sizes.     

COPII under the microscope

Transport through the secretory pathway begins with COPII regulation of ER export. Driven by the Sar1 GTPase cycle, cytosolic COPII proteins exchange on and off the membrane at specific sites on the ER to regulate cargo exit. Here recent developments in COPII research are discussed, particularly the use of live-cell imaging, which has revealed surprising insights into the coat's role. The seemingly static ER exit sites are in fact highly dynamic, and the ability to visualise trafficking processes in intact living cells has highlighted the adaptable nature of COPII in cargo transport and the emerging roles of auxiliary factors.     

Lysophospholipids Facilitate COPII Vesicle Formation

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The structure of a COPII tubule

Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through the secretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500–900 Å vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilize the COPII proteins for transport, but it is unclear how the COPII coat can accommodate these ～3000 Å long molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubule that is approximately 3300 Å long containing a hollow cylindrical interior that is 300 Å in diameter, dimensions that are consistent with those that are required to encapsulate a procollagen molecule wrapped in a membrane and accessory COPII components. This structure suggests a novel mechanism that the COPII coat may employ to transport elongated cargo.     

Molecular mechanisms of COPII vesicle formation

The first step in protein secretion from eukaryotic cells is mediated by COPII vesicles, known for the cytoplasmic coat proteins that are the minimal machinery required to generate these small transport carriers. The five COPII coat components coordinate to create a vesicle by locally generating membrane curvature and populating the incipient bud with the appropriate cargo. This review describes the molecular details of how the COPII coat sculpts vesicles from the endoplasmic reticulum and highlights some unresolved questions regarding the regulation of this process in the complex environment of the eukaryotic cell.     

SNARE selectivity of the COPII coat

The COPII coat buds transport vesicles from the endoplasmic reticulum that incorporate cargo and SNARE molecules. Here, we show that recognition of the ER-Golgi SNAREs Bet1, Sed5, and Sec22 occurs through three binding sites on the Sec23/24 subcomplex of yeast COPII. The A site binds to the YNNSNPF motif of Sed5. The B site binds to Lxx-L/M-E sequences present in both the Bet1 and Sed5 molecules, as well as to the DxE cargo-sorting signal. A third, spatially distinct site binds to Sec22. COPII selects the free v-SNARE form of Bet1 because the LxxLE sequence is sequestered in the four-helix bundle of the v-/t-SNARE complex. COPII favors Sed5 within the Sed5/Bos1/Sec22 t-SNARE complex because t-SNARE assembly removes autoinhibitory contacts to expose the YNNSNPF motif. The COPII coat seems to be a specific conductor of the fusogenic forms of these SNAREs, suggesting how vesicle fusion specificity may be programmed during budding.     

Exclusion size and material have minimal effects on stream benthic algae and macroinvertebrate colonization within submerged cages

Despite their widespread use in grazer–biofilm studies, stream exclusion cages have inherent physical properties that may alter benthic organism colonization and growth. We used laboratory studies and a field experiment to determine how exclusion cage design (size and material) alters light availability, water velocity, and benthic organism colonization. We measured light reduction by various plastic cage materials and flow boundary layer thickness across a range of exclusion cage sizes in the laboratory. We also deployed multiple exclusion cage designs based on commonly available materials into a second-order stream to assess algae and macroinvertebrate colonization differences among exclusion cages. All plastics reduced some light (190–700 nm wavelengths) and blocked more ultraviolet light than photosynthetically active radiation. Exclusion cage size did not influence flow boundary layer thickness, but larger exclusions tended to have higher velocity at the substrata surface. Despite light and water velocity differences, algal biomass, macroinvertebrate density, and community composition were similar between exclusion cage types. However, algal assemblages outside exclusion cages differed in composition and had higher biomass compared to inside exclusion cages. In terms of algal and macroinvertebrate colonization, plastic exclusion cage size and material appear to be flexible within the sizes tested, but differences can still exist between exclusion cage communities and those within the stream. Overall, artifacts of screened exclusion cages do not appear to introduce large bias in results of grazer–biofilm studies, but efforts to design exclusion cages that better mimic the natural system should continue.     

p38 MAPK regulates COPII recruitment

Here, we investigate regulation of coat protein complex II (COPII) recruitment onto ER export sites in permeabilized cells. In cytosols from nocodazole treated HeLa cells we find COPII loading is inhibited. The stress kinase p38 MAPK is activated in these cytosols and COPII loading can be rescued by depletion of p38 MAPK α or by the p38 MAPK inhibitor (SB203580) but not by inhibition/depletion of cdc2. These observations indicate regulation of the early secretory pathway by p38 MAPK.     

Multifaceted roles of COPII subunits in autophagy

COPII vesicles mediate anterograde ER-Golgi traffic of newly synthesized proteins in nutrient rich conditions. An accumulating body of results indicates that the secretory COPII vesicles can be shifted to the roles in autophagosome formation and selective ER-phagy (autophagy of ER), depending on their specific subunits, in response to environmental stresses. In this mini-review, we summarize and discuss the multifaceted roles of COPII vesicles in autophagy and the underlying molecular mechanisms.     

COPII vesicles get supersized by ubiquitin

Some proteins are too big to fit into conventional COPII-coated vesicles, which raises the question of how large cargo, such as procollagen fibrils, are exported from the endoplasmic reticulum. Jin?et?al. (2012) in Nature now report that the creation of oversized vesicles is facilitated by the ubiquitination of the COPII component Sec31p.     

Three-dimensional structure of a COPII prebudding complex

The coat protein complex II (COPII) catalyzes transport vesicle formation from the endoplasmic reticulum. Crystallographic analysis of a Sec23/24-Sar1 prebudding complex of COPII now provides a molecular view of this GTPase-directed coat assembly mechanism.     

Poliovirus Infection Transiently Increases COPII Vesicle Budding

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication "organelles" of PV (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.     

Proteomic Profiling of Mammalian COPII and COPI Vesicles

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Coordination of COPII vesicle trafficking by Sec23

Coat protein complex II (COPII) is a multi-subunit protein complex responsible for the formation of membrane vesicles at the endoplasmic reticulum. The assembly of this complex on the endoplasmic reticulum membrane needs to be tightly regulated to ensure efficient and specific incorporation of cargo proteins into nascent vesicles. Recent studies of a genetic disease affecting COPII function, and a structural analysis of COPII subunit interactions emphasize the central role of the Sec23 subunit in COPII coat assembly. Similarly, the demonstration that Sec23 interacts physically and functionally with proteins involved in both vesicle tethering and the transport along microtubules indicates that the Sec23 subunit is crucially important in linking COPII vesicle formation to anterograde transport events.     

A structural view of the COPII vesicle coat

The COPII vesicle coat coordinates the budding of transport vesicles from the endoplasmic reticulum in the initial step of the secretory pathway. The coat orchestrates a sequence of events including self-assembly on the membrane, cargo and SNARE molecule selection, and deformation of the membrane into a bud to drive vesicle fission. Recent molecular-level studies have helped to explain how the three components of yeast COPII - Sar1 GTPase, the Sec23/24 subcomplex and the Sec13/31 subcomplex - combine to organize this complex process.     

Mechanisms of COPII vesicle formation and protein sorting

The evolutionarily conserved coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). COPII coat is responsible for direct capture of cargo proteins and for the physical deformation of the ER membrane that drives the COPII vesicle formation. In addition to coat proteins, recent data have indicated that the Ras-like small GTPase Sar1 plays multiple roles, such as COPII coat recruitment, cargo sorting, and completion of the final fission. In the present review, we summarize current knowledge of COPII-mediated vesicle formation from the ER, as well as highlighting non-canonical roles of COPII components.