Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

Abstract

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.

Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport

“…To exit the ER after serum starvation, mammalian Sac1 is thought to oligomerize via its N‐terminal LZ motif and interact with the COPII subunit Sec23 (Figure C). Sac1 incorporation into COPII vesicles requires the cytosolic adaptor protein 14‐3‐3, which directly binds a minimal ER sorting motif in SacN (L146S147) that lies within a 7 amino acid sequence (RLSNTSP) similar to consensus 14‐3‐3 binding sites (Figure D). Because 14‐3‐3 proteins can also bind the COPII sorting subunit Sec24, 14‐3‐3 dimers may act as a bridge to concentrate Sac1 at ER exit sites and facilitate its integration into COPII‐vesicles destined for the Golgi .…”

“…Sac1 incorporation into COPII vesicles requires the cytosolic adaptor protein 14‐3‐3, which directly binds a minimal ER sorting motif in SacN (L146S147) that lies within a 7 amino acid sequence (RLSNTSP) similar to consensus 14‐3‐3 binding sites (Figure D). Because 14‐3‐3 proteins can also bind the COPII sorting subunit Sec24, 14‐3‐3 dimers may act as a bridge to concentrate Sac1 at ER exit sites and facilitate its integration into COPII‐vesicles destined for the Golgi . For Golgi exit and transport back to the ER following serum stimulation, a dilysine motif (KEKIDD) at the Sac1 C‐terminus is required for COPI‐vesicle transport (Figures C and C).…”

Silencing of Small Valosin-containing Protein-interacting Protein (SVIP) Reduces Very Low Density Lipoprotein (VLDL) Secretion from Rat Hepatocytes by Disrupting Its Endoplasmic Reticulum (ER)-to-Golgi Trafficking

“…These proteins not only facilitate the selection of their specific cargos into different vesicles, but they also act as adaptors of cargo proteins, allowing interaction with COPII coat proteins and modulating the size of transport vesicles. Recently, 14-3-3, a phosphoregulatory protein, has been shown to act as an adaptor protein for a phosphatidylinositol 4-lipid phosphate, SAC1 (24). SAC1 exits the ER in a COPII-dependent manner; however, its efficient packaging into COPII vesicles requires 14-3-3 protein (24).…”

“…Recently, 14-3-3, a phosphoregulatory protein, has been shown to act as an adaptor protein for a phosphatidylinositol 4-lipid phosphate, SAC1 (24). SAC1 exits the ER in a COPII-dependent manner; however, its efficient packaging into COPII vesicles requires 14-3-3 protein (24). Utilizing a variety of cell biological and biochemical techniques, it has been shown that the export .…”

Emerging themes of regulation at the Golgi

“…In growth factor-deprived cells, SAC1 oligomerizes and traffics from the ER to the Golgi, depleting Golgi PtdIns(4)P, thus interfering with secretory trafficking [9]. Since SAC1 is exported from the ER into COPII vesicles at similar levels in starved and non-starved cells, growth factor-dependent ERGolgi trafficking of SAC1 is likely regulated at the level of Golgi retrieval [10]. Stimulation with growth factors such as FGF and PDGF activates the p38/MAPK pathway, triggering dissociation of SAC1 oligomers and COPI-mediated retrograde trafficking of SAC1 to the ER.…”