Transposon-Disruption of a Maize Nuclear Gene, tha1, Encoding a Chloroplast SecA Homologue: In Vivo Role of cp-SecA in Thylakoid Protein Targeting

Abstract

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced ≥40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

The Maize tha4 Gene Functions in Sec-Independent Protein Transport in Chloroplasts and Is Related to hcf106, tatA, and tatB

“…Total DNA was extracted from maize seedlings and analyzed by Southern hybridization with digoxigenin-labeled Mu probes, as described previously (Voelker et al, 1997). Total leaf RNA was extracted with TRIzol Reagent (GIBCO BRL) according to the manufacturer's instructions.…”

“…Because the amount of tha9 -specific cDNA sequence was insufficient to generate a sensitive gene-specific probe for RNA gel blots ( ‫ف‬ 40 nucleotides), RNase protection assays were used to distinguish the tha4 and tha9 mRNAs in different maize tissues. RNase protection assays were performed as described in Voelker et al (1997). The radioactive antisense probes were generated from near full-length tha4 and tha9 cDNA sequences.…”

Mechanisms of Protein Import and Routing in Chloroplasts

“…Maize mutants deficient in SecY or SecA have also been characterized in some detail [91,92]. Substrates for this system bear Sec-type signal peptides broadly similar to bacterial signal peptides: the peptides have a characteristic three-domain structure that comprises a positively charged amino-terminal region (N-domain), hydrophobic core region (H-domain) and more polar carboxy-terminal region (C-domain) ending with an Ala-Xaa-Ala consensus sequence recognized by the thylakoidal processing peptidase (TPP).…”

Proteomic identification of the interactome of stalled ribosome nascent chain complexes translating the thylakoid membrane protein D1

“…CpSRP54 is also able to contact the nascent chain of D1 after its emergence from the ribosomal exit tunnel (Nilsson et al, 1999;Nilsson and van Wijk, 2002). An alternative cotranslational targeting mechanism was described for the chloroplast-encoded cytochrome b6f subunit cytochrome f. Cytochrome f is synthesized with a cleavable N-terminal signal peptide and is targeted by chloroplast SecA (cpSecA), a homologue of the bacterial ATPase SecA, which mediates targeting of preproteins to the plasma membrane Sec machinery for translocation (Voelker and Barkan, 1995;Voelker et al, 1997;Röhl and van Wijk, 2001;Zoschke and Barkan, 2015;Fernandez, 2018). Consistently, cpSecA was identified in the ribosome-associated proteome of Chlamydomonas chloroplasts (Westrich et al, 2021).…”