Examples of long coil-coiled tethering factors include the golgin family of proteins at the Golgi apparatus, and EEA1 on the endosomes 11, 13. Long coil-coiled tethers tend to be large (>60 kDa) and form a coiled-coil domain structure. The initial ‘capture’ with the tether is therefore essential for correct vesicle targeting and fidelity of cargo delivery. Molecular tethers either long coil-coiled tethers or multisubunit tethering complexes interact with the carrier prior to the subsequent SNARE-mediated fusion 11, 12. The fusion of intracellular carriers is understood to be a two-step process. These carriers are then trafficked directly to the plasma membrane where they fuse and their contents are delivered to the extracellular milieu 2, 10. At the trans-Golgi apparatus, soluble secreted proteins are sorted into pleomorphic post-Golgi tubular carriers 2. After proper folding, they are trafficked to the Golgi apparatus in COPII carriers where they are glycosylated 1, 6– 9. Soluble secreted proteins are synthesised in the endoplasmic reticulum. In higher eukaryotes, approximately 12% of all proteins are secreted from the cell 3– 5 where they fulfil a vast array of different functions, including cell signalling, the immune response, and extracellular matrix (ECM) remodelling 2. Proteins are transported from their site of synthesis in the endoplasmic reticulum to the Golgi apparatus where they are sorted to different subcellular localisations, such as the endolysosomal system or directly to the plasma membrane for secretion 1, 2. The complex process of membrane trafficking is fundamental to cellular organisation. Together these data identify the exocyst complex as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway. Importantly, the knock-out of exocyst subunits in specialised secretory cell types prevents the constitutive secretion of antibodies in lymphocytes and of the hormones leptin and adiponectin in adipocytes. Unbiased secretomics reveals a drastic reduction in the secretion of soluble proteins to the extracellular milieu after knock-out of exocyst subunits. Abrogation of exocyst followed by kinetic trafficking assays with multiple different soluble cargoes results in cargo accumulation of all tested cargoes. Exocyst subunits co-localise with carriers fusing at the plasma membrane and we show that the exocyst complex is recruited directly to these carriers. Depletion of core-exocyst subunits reduces carrier fusion and causes cargo accumulation in the post-Golgi carriers.
Here, we have used kinetic trafficking assays and transient CRISPR knock-outs to study the biosynthetic sorting route from the Golgi apparatus to the plasma membrane. The molecular machinery involved is poorly understood. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane to deliver their contents to the extracellular space. Secreted proteins fulfil a vast array of different functions, including adaptive immunity, cell signalling and extracellular matrix remodelling.
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