Abstract
BackgroundNonclassical (unconventional) protein secretion is thought to represent the primary secretion mechanism for several cytosolic proteins, such as HIV-Tat, galectin 1, interleukin-1β, and several proteins that shuttle between the nucleus and cytosol, such as fibroblast growth factor 1 (FGF1), FGF2, and nucleolin. Four nonclassical secretory pathways have been described including direct transport (presumably through transporters in the plasma membrane), secretion via exosomes, lysosomal secretion, and blebbing. The purpose of this study was to gain mechanistic insight into nonclassical protein secretion using phosphoglycerate kinase 1 (PGK1), a previously identified nonclassical secretory protein, as a reporter protein.ResultsUpon shifting HeLa cells into serum-free media PGK1 was released as a free soluble protein without cell loss. Release occurred in two phases: a rapid early phase and a slow late phase. Using a repertory of inhibitors, PGK1 release was shown not to rely on the classical secretory pathway. However, components of the cytoskeleton partially contributed to its release. Significantly, the presence of serum or bovine serum albumin in the media inhibited PGK1 release.ConclusionsThese results are consistent with a novel model of protein release termed oncotic release, in which a change in the colloidal osmotic pressure (oncotic pressure) upon serum withdrawal creates nonlethal oncotic pores in the plasma membrane through which PGK1 - and likely other nearby proteins - are released before the pores are rapidly resealed. These findings identify an alternative mechanism of release for FGF1, HIV-Tat, and galectin 1 whose reported nonclassical secretion is induced by serum withdrawal. Oncotic release may occur in routine cell biological experiments during which cells are washed with serum-free buffers or media and in pathophysiological conditions, such as edema, during which extracellular protein concentrations change.
Highlights
Nonclassical protein secretion is thought to represent the primary secretion mechanism for several cytosolic proteins, such as HIV-Tat, galectin 1, interleukin-1b, and several proteins that shuttle between the nucleus and cytosol, such as fibroblast growth factor 1 (FGF1), FGF2, and nucleolin
phosphoglycerate kinase 1 (PGK1) as reporter for nonclassical secretion Hogg and coworkers previously demonstrated that PGK1 can be released from a variety of cells lines including HT1080 cells [9]
HeLa cells were used as a model system, because they are routinely used in the laboratory and the amount of PGK1 released from them [11] and HT1080 cells [9] were comparable
Summary
Nonclassical (unconventional) protein secretion is thought to represent the primary secretion mechanism for several cytosolic proteins, such as HIV-Tat, galectin 1, interleukin-1b, and several proteins that shuttle between the nucleus and cytosol, such as fibroblast growth factor 1 (FGF1), FGF2, and nucleolin. Four nonclassical secretory pathways have been described including direct transport (presumably through transporters in the plasma membrane), secretion via exosomes, lysosomal secretion, and blebbing. The purpose of this study was to gain mechanistic insight into nonclassical protein secretion using phosphoglycerate kinase 1 (PGK1), a previously identified nonclassical secretory protein, as a reporter protein Several important proteins, such fibroblast growth factor 1 (FGF1), FGF2, and interleukin-1b (IL-1b) are secreted from cells by alternative pathways collectively termed nonclassical (unconventional) secretory pathways [1]. Four nonclassical protein secretory pathways have been described (reviewed in [2]) They include 1) direct transport of proteins from the cytosol across the plasma membrane presumably through membrane transporters, 2) lysosomal secretion, 3) export via exosomes derived from multivesicular bodies, and 4) packaging of proteins into plasma membrane vesicles (blebbing). IL-1b can be exported in secretory lysosomes [5], blebs [6], exosomes [7], or directly through the plasma membrane by unknown transporters [8]
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