Juneau Andrea D, Frankel Laurie K, Bricker Terry M, Roose Johnna L
Department of Biological Sciences, Biochemistry and Molecular Biology Section, Louisiana State University, Baton Rouge, Louisiana, 70803, United States of America.
PLoS One. 2016 Sep 22;11(9):e0163646. doi: 10.1371/journal.pone.0163646. eCollection 2016.
The CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.
氰基Q蛋白已被证明是蓝藻光系统II（PS II）的一个组成部分，但关于它与该复合物的物理关联仍存在许多悬而未决的问题。氰基Q是一种脂蛋白；在信号肽酶II切割其转运肽后，成熟蛋白被递送至类囊体腔空间，其N端半胱氨酸残基会发生脂质修饰。这种修饰似乎将这个原本可溶的组分拴在了类囊体膜上。为了探究脂质锚定的功能意义，已在集胞藻PCC 6803中构建了氰基Q蛋白的突变体，以消除N端半胱氨酸残基，从而阻止脂质修饰。用丝氨酸取代N端半胱氨酸残基（Q-C22S）导致可检测到的氰基Q蛋白量降至野生型蛋白的17%。此外，积累的Q-C22S蛋白的物理性质与氰基Q前体加工过程的改变一致。在TX-114相分配实验中，Q-C22S蛋白迁移至更高的表观分子量并分配到疏水相中。这些结果表明，疏水的N端22个氨基酸未被信号肽酶正确切割。用可溶性腔蛋白PsbO的转运肽取代整个氰基Q转运肽产生了Q-SS突变体，且未检测到修饰后的氰基Q蛋白积累。最后，在PS II突变体菌株ΔpsbB和ΔpsbO中，氰基Q蛋白含量正常，这表明与PS II的关联并非氰基Q稳定积累的先决条件。这些结果共同表明，集胞藻PCC 6803中氰基Q的积累取决于N端脂质锚定的存在，而不取决于氰基Q与PS II复合物的关联。
