Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis.
The Arabidopsis genome contains 14 genes encoding the serine protease DegP. Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane. We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically. Size-exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers. Proteolytic activity was characterized using beta-casein as a model substrate. DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures. DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen. The results of this work are discussed in the context of the recent elucidation of the structure of the E. coli homolog and the possible physiological role of the protease in the chloroplast lumen.
Chassin
Yael
Y
Institute of Plant Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Kapri-Pardes
Einat
E
Sinvany
Galit
G
Arad
Tal
T
Adam
Zach
Z
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D010970:Plastocyanin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D013696:Temperature; D020524:Thylakoids / Q000201:enzymology*
D029681:Arabidopsis Proteins; D002364:Caseins; D011993:Recombinant Fusion Proteins; D010970:Plastocyanin; D010450:Endopeptidases; D012697:Serine Endopeptidases