Abstract
The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed us to identify at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes.
Highlights
Constitutive secretion delivers newly synthesised proteins and lipids to the cell surface and is essential for cell growth and viability
The majority of secreted proteins begin their journey at the endoplasmic reticulum, pass through the Golgi, and are transported to the cell surface in small vesicles/tubules which fuse with the plasma membrane
The molecular understanding of this fusion step is still unclear and in higher eukaryotes it is not known which SNARE proteins drive this process. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with gene depletion studies in Drosophila cells. Using this assay we identified three SNARE complexes driving the fusion of secretory vesicles with the plasma membrane and uncovered an unexpected role for the RSNARE YKT6 in this process
Summary
Constitutive secretion delivers newly synthesised proteins and lipids to the cell surface and is essential for cell growth and viability. This pathway is required for the exocytosis of molecules such as antibodies, cytokines and extracellular matrix components so has both significant physiological and commercial importance. The majority of constitutive secreted proteins are synthesised at the endoplasmic reticulum, pass through the Golgi, and are transported to the cell surface in small vesicles and tubules which fuse with the plasma membrane [1, 2]. In some cell types, such as MDCK cells and macrophages, there is evidence that constitutive secretory cargo passes through a endosomal intermediate on its way to the cell surface [5, 6]. In non-polarised cells endosomal intermediates do not appear to play a major role in this pathway [7]
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