RESUMO
Since the first fluorescent proteins (FPs) were identified and isolated over fifty years ago, FPs have become commonplace yet indispensable tools for studying the constitutive secretory pathway in live cells. At the same time, genetically encoded chemical tags have provided a new use for much older fluorescent dyes. Innovation has also produced several specialized methods to allow synchronous release of cargo proteins from the endoplasmic reticulum (ER), enabling precise characterization of sequential trafficking steps in the secretory pathway. Without the constant innovation of the researchers who design these tools to control, image, and quantitate protein secretion, major discoveries about ER-to-Golgi transport and later stages of the constitutive secretory pathway would not have been possible. We review many of the tools and tricks, some 25 years old and others brand new, that have been successfully implemented to study ER-to-Golgi transport in intact and living cells.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Transporte ProteicoRESUMO
ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by â¼50%. We found that at steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.