Proteolytic processing of thylakoid proteins

Nuclear-encoded thylakoid proteins are targeted into and across the thylakoid membrane by four distinct mechanisms. Precursors of lumenal proteins are translocated by either a Sec- or ΔpH-dependent mechanism. In both cases, thylakoidal processing peptidase (TPP) removes this signal peptide to release the mature protein. A structurally similar signal peptide is present in the fourth pathway used by a subset of integral membrane proteins including PSII-W, PSII-X and PsbY. In this case the integration process does not involve an identifiable energy source or known protein transport machinery. This Sec-independent direct insertion mechanism is unlike any other known, with the exception of a single protein, M13 procoat, which similarly inserts in the Escherichia coli plasma membrane by means of a signal peptide. This novel insertion mechanism has been probed by mutagenesis of TPP cleavage sites within pre-PSII-W, -PSII-X and -PsbY, in order to generate intermediates on the insertion pathway. TPP cleaves preferentially after Ala-X-Ala, and the terminal alanine was mutated to threonine by site-specific mutagenesis of the cDNA. Chapter 3 - In the case of pre-PSII-W and pre-PSII-X, TPP is the only proteinaceous component known to be involved at any stage of the insertion process. The PSII-W cleavage site mutant is imported and inserted into the thylakoid membrane, but cleavage by TPP is inhibited. Import into chloroplasts results in the accumulation of a mature size protein and an intermediate form that accumulates when TPP fails to complete the maturation (the precursor protein is processed to an intermediate in the stroma). Importantly, the intermediate is located exclusively in the thylakoid membrane, confirming that the action of TPP is not required for correct localisation. Protease-topology mapping presented, shows that this intermediate is in the form of a loop intermediate in which both the N- and C-termini are exposed on the stromal face of the membrane with the intervening region on the lumenal face. Preliminary data is also presented for a loop intermediate with the insertion of pre-PSII-X. Chapter 4 - Precursor PsbY has a more complex insertion mechanism and 6 mutants are presented that show various intermediates on the pre-PsbY insertion pathway. All the intermediates are stable in the thylakoid membrane. Furthermore, using this approach, it can be shown that pre-PsbY is in fact a polyprotein containing two similar single-span proteins, both of which are preceded by cleavable signal peptides. Pre-PsbY thus contains a single chloroplast targeting peptide, an initial thylakoid signal peptide and mature protein followed by a second thylakoid signal peptide and mature protein. This is therefore the first nuclear-encoded polyprotein targeted to the thylakoid membrane in higher plants, and the data suggest that this protein inserts as a double-loop form