Binding of Proteolytically Processed Phospholipase D from Streptomyces chromofuscus to Phosphatidylcholine Membranes Facilitates Vesicle Aggregation and Fusion

• Published in: Biochemistry (Easton) 2001-11, Vol.40 (46), p.13954-13963
• Main Authors: Stieglitz, Kimberly A, Seaton, Barbara A, Roberts, Mary F
• Format: Article
• Language: English
• Subjects: 4-Chloro-7-nitrobenzofurazan - analogs & derivatives; 4-Chloro-7-nitrobenzofurazan - chemistry; 4-Chloro-7-nitrobenzofurazan - metabolism; Barium - chemistry; Barium - metabolism; Calcium - chemistry; Calcium - metabolism; Cryoelectron Microscopy; Endopeptidases - metabolism; Energy Transfer; Hydrolysis; Ligands; Light; Liposomes - chemistry; Liposomes - metabolism; Membrane Fusion; Phosphatidic Acids - chemistry; Phosphatidic Acids - metabolism; Phosphatidylcholines - chemistry; Phosphatidylcholines - metabolism; Phospholipase D - biosynthesis; Phospholipase D - chemistry; Phospholipase D - metabolism; Protein Binding; Protein Processing, Post-Translational; Scattering, Radiation; Spectrometry, Fluorescence; Streptomyces - enzymology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi011338o

Ca2+-dependent phospholipase D is secreted from Streptomyces chromofuscus as an intact enzyme of 57 kDa (PLD57). Under certain growth conditions, PLD is proteolytically cleaved and activated to form PLD42/20 (named for the apparent size of the peptides). The PLD42 catalytic core and 20 kDa C-terminal domain remain tightly associated through noncovalent interactions. In the presence of Ba2+ (to enhance protein binding to zwitterionic vesicles without hydrolysis of substrate), PLD42/20, but not PLD57, induces POPC vesicle leakiness as measured by entrapped CF leakage. PLD42/20 also induces vesicle fusion (as measured by light scattering, fluorescence quenching, and cryo-TEM) under these conditions (1 mM POPC, 5 mM Ba2+); neither PLD42 nor PLD20 alone can act as a fusogen. For intact PLD57 to cause CF leakiness, the soluble activator diC4PA must be present. However, even with diC4PA, PLD57 does not induce significant vesicle fusion. In the absence of metal ions, all PLD forms bind to PC vesicles doped with 10 mol % PA. Again, only PLD42/20 is fusogenic and causes aggregation and fusion on a rapid time scale. Taken together, these data suggest that activated PLD42/20 inserts more readily into the lipid bilayer than other PLD forms and creates structures that allow bilayers to fuse. Cleavage of the PLD57 by a secreted protease to generate PLD42/20 occurs in the late stages of S. chromofuscus cell cultures. Production of this more active and fusogenic enzyme may play a role in nutrient scavenging in stationary phase cultures.