An AAA protease FtsH can initiate proteolysis from internal sites of a model substrate, apo-flavodoxin.
Escherichia coli FtsH, which belongs to the AAA (ATPases associated with diverse cellular activities) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. It has been proposed that ATP-dependent proteases unfold substrate proteins and initiate a processive proteolysis from either terminus of the substrate polypeptide. We have found that FtsH degrades E. coli apo-flavodoxin (apo-Fld) but not holo-Fld containing non-covalently bound flavin mononucleotide (FMN). A mutant Fld carrying a substitution of Tyr94 to Asp (Fld(YD)) with a lower affinity for FMN was efficiently degraded by FtsH. To elucidate the directionality of Fld(YD) degradation by FtsH, we constructed several Fld(YD) fusion proteins with glutathione S-transferase (GST), green fluorescent protein (GFP), or both GST and GFP. It was found that FtsH was able to initiate degradation of the Fld(YD) moiety even when it was sandwiched by GST and GFP. Evidence indicated that FtsH can initiate proteolysis of GST-Fld(YD)-GFP from the Fld(YD) moiety by translocating an internal loop to the protease chamber in an ATP-dependent manner and that, at least, the proteolysis in the C to N direction proceeds processively.
Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
Takashi
T
Okuno
Kunitoshi
K
Yamanaka
Teru
T
Ogura
D049069:ATP-Dependent Proteases; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005486:Flavin Mononucleotide / Q000378:metabolism; D005418:Flavodoxin / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005486:Flavin Mononucleotide; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D010447:Peptide Hydrolases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli