Abstract
Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.
Highlights
Perforin is a pore-forming protein that delivers granzymes to eliminate compromised cells
In this study we have conclusively shown that perforin is able to deliver molecules other than granzymes into target cells
What is the explanation for this charge-based facilitated diffusion? We originally hypothesized that granzyme delivery by perforin involves electrostatic interaction at the plasma mem
Summary
Perforin is a pore-forming protein that delivers granzymes to eliminate compromised cells. We demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently Another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), favors the delivery of cationic molecules, and efficiently delivers human granzyme B. There are multiple lines of evidence that suggest perforin forms a pore at the plasma membrane to allow access of granzymes into the target cell These include images of pores on the target cell membrane after a killer cell attack [11]; the finding that the calcium influx after treatment with perforin only lasts for 180 s [12]; and perforinmediated delivery and release of various molecules including fluorescein isothiocyanate (FITC)-labeled dextrans, up to 20 kDa in size, into resealed human erythrocyte ghosts and giant unilamellar vesicles [13,14,15]. We conclude that physiologically relevant perforin cargos have evolved to enter the cell via a process of facilitated diffusion through the perforin pore
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