The COPII complex mediates the selective incorporation of secretory cargo and relevant machinery into budding vesicles at specialised sites on the endoplasmic reticulum membrane called transitional ER (tER). Here, we show using confocal microscopy, immunogold labelling of ultrathin cryosections and electron tomography that in human cells at steady state, Sec16 localises to cup-like structures of tER that are spatially distinct from the localisation of other COPII coat components. We show that Sec16 defines the tER, whereas Sec23-Sec24 and Sec13-Sec31 define later structures that precede but are distinct from the intermediate compartment. Steady-state localisation of Sec16 is independent of the localisation of downstream COPII components Sec23-Sec24 and Sec13-Sec31. Sec16 cycles on and off the membrane at a slower rate than other COPII components with a greater immobile fraction. We define the region of Sec16A that dictates its robust localisation of tER membranes and find that this requires both a highly charged region as well as a central domain that shows high sequence identity between species. The central conserved domain of Sec16 binds to Sec13 linking tER membrane localisation with COPII vesicle formation. These data are consistent with a model where Sec16 acts as a platform for COPII assembly at ERES.

The COPII coat assembles on endoplasmic reticulum membranes to coordinate the collection of secretory cargo with the formation of transport vesicles. During COPII assembly, Sar1 deforms the membrane and recruits the Sec23-Sec24 complex (Sec23/24), which is the primary cargo-binding adaptor for the system, and Sec13-Sec31 (Sec13/31), which provides a structural outer layer for vesicle formation. Here we show that Sec13 depletion results in concomitant loss of Sec31 and juxtanuclear clustering of pre-budding complexes containing Sec23/24 and cargo. Electron microscopy reveals the presence of curved coated profiles on distended endoplasmic reticulum, indicating that Sec13/31 is not required for the generation or maintenance of the curvature. Surprisingly, export of tsO45-G-YFP, a marker of secretory cargo, is unaffected by Sec13/31 depletion; by contrast, secretion of collagen from primary fibroblasts is strongly inhibited. Suppression of Sec13 expression in zebrafish causes defects in proteoglycan deposition and skeletal abnormalities that are grossly similar to the craniofacial abnormalities of crusher mutant zebrafish and patients with cranio-lenticulo-sutural dysplasia. We conclude that efficient coupling of the inner (Sec23/24) and outer (Sec13/31) layers of the COPII coat is required to drive the export of collagen from the endoplasmic reticulum, and that highly efficient COPII assembly is essential for normal craniofacial development during embryogenesis.

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKC␣, is a major regulator of platelet granule secretion, but the molecular pathway from PKC␣ to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in ␣-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion. (Blood. 2011; 118(2):416-424) IntroductionPlatelet activation underlies the arterial thrombosis that causes the acute severe symptoms of heart disease and thrombotic stroke, 1 and it is therefore important to determine the molecular mechanisms regulating platelet activity. We and others have shown that protein kinase C (PKC) isoforms regulate all of the essential functions of platelets, including actin rearrangements, adhesion through integrins, and secretion of granule contents. [2][3][4][5][6] Of the isoforms of PKC expressed in platelets, the conventional PKCs, PKC␣ and PKC␤, have clear positive signaling roles, and mouse platelets lacking expression of PKC␣ show marked attenuation of responses and thrombus formation. 2,7 The critical function regulated by PKC␣ is the secretion of dense granule content, which is rescued by the addition of exogenous ADP. 2 Therefore, we sought to identify proteins that lie downstream of PKC in the pathway to regulation of dense-granule secretion to investigate the molecular regulation of this essential function.The protein kinase D (PKD) family of Ser/Thr kinases consists of 3 members, PKD1 (also known as PKC), PKD2 and PKD3. 8 PKDs contain a tandem repeat of zinc finger-like cysteine-rich motifs at their N-termini, highly homologous to domains found in diacylglycerol (DAG)/phorbol ester-sensitive PKCs and other signaling proteins regulated by DAG. However, unlike PKCs, PKDs lack the C2 domain responsible for the Ca 2ϩ sensitivity of conventional PKCs, whereas they possess an autoinhibitory PH domain. Further, the catalytic domain of PKD has low homology with the conserved kinase domain of the PKCs. These differences make the PKD family a distinct set of ki...

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