Component Specificity for the Thylakoidal Sec and Delta Ph–Dependent Protein Transport Pathways

Abstract

Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in ∼10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

Functional assembly of thylakoid ΔpH‐dependent/Tat protein transport pathway components in vitro

“…As reported previously [10,14], in vitro translated pHcf106 and pTha4 are imported into intact chloroplasts, processed to mature size, and integrated into thylakoids (Fig. 1A, lanes 1-7).…”

“…Antibodies to pea Hcf106, Tha4, cpTatC, cpSecY and cpOxa1p have been described [8,10,14]. Antibodies to maize Hcf106 were as described [14] and antibodies to maize Tha4 were the generous gift of A. Barkan [15].…”

“…Mutant tha4/tha4 maize seedlings were selected by their pale green phenotype and by high chlorophyll fluorescence with a hand-held UV lamp. Maize chloroplasts were isolated as described [14]. Chloroplast lysate, washed thylakoids and stromal extract were prepared from isolated chloroplasts [18].…”

“…An altered form of mHcf106 (mHcf106 E 11 Q) was derived by PCR amplification using a 5¢ primer that mutated nucleotides encoding glutamate 11 of the engineered mHcf106 to glutamine. The mature form of pea Tha4 (mTha4) was cloned by PCR amplification from pTha4 [14] based on the predicted transit peptide cleavage site from a combination of ChloroP [16] and alignment with orthologous proteins. The 5¢ primer (including an engineered KpnI site) was used to mutate the nucleotides encoding asparagine 56 to encode methionine such that the resulting protein started MAFFGLGVPELVV…; the 3¢ primer bound in the pGEM 4Z vector.…”

Plant mitochondria contain the protein translocase subunits TatB and TatC

“…Thus, we sought to identify any possible TatA-or TatBencoding genes in the Arabidopsis genome that might be targeted to mitochondria. We first excluded the chloroplast-targeted cpTatA and cpTatB as being dual-targeted to mitochondria, by using antibodies to the Pisum sativum ( pea) chloroplast TatA (Mori et al, 1999) and TatB proteins (hereafter cpTatA and cpTatB) on isolated chloroplastic and mitochondrial fractions from pea (Fig. 1C).…”