Protein release through nonlethal oncotic pores as an alternative nonclassical secretory pathway.
BACKGROUND
Nonclassical (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.


RESULTS
Upon 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.


CONCLUSIONS
These 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.
Department of Cell Biology and Molecular & Cellular Biology Program, State University of New York Downstate Medical Center, Brooklyn, 11203, USA. william.chirico@downstate.edu
William J
WJ
Chirico
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002452:Cell Count; D016895:Culture Media, Serum-Free; D015638:Cytochalasin D / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008954:Models, Biological; D009997:Osmotic Pressure; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013997:Time Factors
D016895:Culture Media, Serum-Free; D015638:Cytochalasin D; D012710:Serum Albumin, Bovine; D010735:Phosphoglycerate Kinase