Lohman, T. M. | Search bioRxiv

bioRxiv2020

Comparative analysis of CPI-motif regulation of biochemical functions of actin capping protein

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs(1, 2) across all protein families (1, 2). These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

biochemistry↗

bioRxiv2019

A Novel Antenna Protein Complex in the Life Cycle of Cyanobacterial Photosystem II

In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. However, many facets of its repair and reassembly following photodamage remain unknown. We have discovered a novel PSII subcomplex that lacks five key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment-protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low-molecular-mass subunits, and the assembly factor Psb27. Immunoblotting analysis, multiple mass spectrometry techniques, and ultrafast spectroscopic results supported the absence of a functional reaction center in this chlorophyll-protein complex. We therefore refer to it as the no reaction center complex (NRC). Additionally, genetic deletion of PsbO on the PSII lumenal side resulted in an increased NRC population, indicative of a faulty PSII repair scheme at the cellular level. Analytical ultracentrifugation studies and clear native acrylamide gel analysis showed that the NRC complex is a stable pigment-protein complex and not a mixture of free CP47 and CP43 proteins. Our finding challenges the current model of the PSII repair cycle and implies an alternative PSII repair strategy. We propose that formation of this pigment-protein complex maximizes PSII repair economy by preserving an intact PSII core antenna shell in a single complex that is available for PSII reassembly, thus minimizing the risk of randomly diluting multiple recycling components in the thylakoid membrane following a photodamage event at the RC.\n\nSignificance statementPhotosystem II (PSII) converts sunlight into chemical energy, powering nearly all life on Earth. The efficiency of this process is maximized under various environmental conditions by a frequent repair and reassembly cycle that follows inevitable PSII damage even during normal oxygenic photosynthesis. We have isolated a novel pigment protein PSII subcomplex in which, surprisingly, the reaction center (RC) components of PSII are absent. Formation of this stable chlorophyll-protein complex suggests a protective mechanism whereby longer-lived PSII subunits are unplugged from the damaged RC to prevent harmful, aberrant photochemistry during RC repair. This finding provides intriguing new insight into how PSII is assembled and rebuilt to optimize its performance to optimally catalyze one of the most challenging reactions in biology.

plant biology↗