Abstract
Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.
Highlights
Complexes of SNAREs from opposing membranes yields a close, stable proximity between the two membranes, which facilitates overcoming the energy barrier required for membrane fusion [3,4,5]
Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells
Secretion Is Inhibited in Response to Amino Acid Deprivation—Autophagy triggered by starvation may be regarded as a special case of membrane-trafficking process that shares some of the components utilized by other intracellular membranetrafficking pathways
Summary
Complexes of SNAREs from opposing membranes yields a close, stable proximity between the two membranes, which facilitates overcoming the energy barrier required for membrane fusion [3,4,5]. The coupled antibodies were incubated with either recombinant His-NSF-Myc protein (0.5 g) or cytosolic (200 g) and membrane extract fractions (200 g) obtained from control or starved cells for at least 2 h at 4 °C.
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