Hd. Tang et Pr. Chitnis, Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I, I J BIOCH B, 37(6), 2000, pp. 433-440
In vitro mutagenesis was used to produce two photosystem I mutants of the c
yanobacterium Synechocystis sp. PCC 6803. The mutants HK and HL contained h
exahistidyl tags at the C-termini of the PsaK1 and PsaL subunits, respectiv
ely. The HK mutant contained wild-type amounts of trimeric PS I complexes,
but the level of hexahistidine-tagged PsaK1 was found only ten per cent in
the PS I complexes and membranes of the wild type level. Therefore, attachm
ent of a tag at the C-terminus interferes with the expression or assembly o
f PsaK1. In contrast, the HL mutant contained a similar level of tagged Psa
L as that in the wild type. However, trimeric PS I complexes could not be o
btained from this strain, indicating that the C-terminus of PsaL is involve
d in the formation of PS I trimers. Hexahistidine-tagged complexes of the H
L and HK strains could not be purified with Nickel-affinity chromatography,
unless photosystem I was denatured with urea, demonstrating that tagged C-
termini of PsaK1 and PsaL were embedded inside of the PS I complex. Protect
ion of the C-terminus from trypsin cleavage further supported this conclusi
on. Thus, histidine tagging allowed us to demonstrate role of C-termini of
two proteins of photosystem I.