The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog

Abstract

The known envelope membrane proteins of the chloroplastic protein import apparatus lack sequence similarity to proteins of other eukaryotic or prokaryotic protein transport systems. However, we detected a putative homolog of the gene encoding Toc75, the protein-translocating channel from the outer envelope membrane of pea chloroplasts, in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We investigated whether the low sequence identity of 21% reflects a structural and functional relationship between the two proteins. We provide evidence that the cyanobacterial protein is also localized in the outer membrane. From this information and the similarity of the predicted secondary structures, we conclude that Toc75 and the cyanobacterial protein, referred to as SynToc75, are structural homologs. synToc75 is essential, as homozygous null mutants were not recovered after directed mutagenesis. Sequence analysis indicates that SynToc75 belongs to a family of outer membrane proteins from Gram-negative bacteria whose function is not yet known. However, we demonstrate that these proteins are related to a specific group of prokaryotic secretion channels that transfer virulence factors, such as hemolysins and adhesins, across the outer membrane.

Figure 1

Schematic representation of the sequence alignment between pea Toc75 and SynToc75 (Slr1227) from Synechocystis sp. PCC 6803. The degree of sequence similarity between certain segments of the proteins is indicated by gray shading.

Figure 2

Localization of SynToc75 (Slr1227) in the outer membrane of Synechocystis sp. PCC 6803. After cell disruption, the cell components were fractionated either by ultracentrifugation into soluble (S) and insoluble (IS) fractions or by sucrose density gradients (20) into remaining whole cells (WC), plasma membranes (PM), thylakoids (THY), and outer membranes (OM). The outer membrane fraction was incubated in 1.0 M NaCl or 0.1 M Na₂CO₃, pH 11.5, and soluble (S) and pellet fractions (P) were separated by ultracentrifugation. Proteins in each fraction were analyzed by SDS/PAGE and immunoblotting. SomA and PsaD were used as marker proteins for outer and thylakoid membranes, respectively.

Figure 3

Generation of synToc75 (slr1227) mutants. (A) The wild-type gene synToc75 (slr1227) of Synechocystis sp. PCC 6803 was disrupted either by an insertion of the kanamycin-resistance gene, kan^r, into synToc75 (synToc75-kan^r) or by a replacement of the synToc75 ORF by the kan^r ORF (ΔsynToc75-kan^rORF). Only the data obtained for the sense orientation of the kan^r insertion are shown (synToc75-kan^r). (B) Segregation of wild-type synToc75 was analyzed by DNA gel blotting. Transformed cells were grown under photoautotrophic conditions and transferred to new plates for the number of passages indicated. (C) As a control for genome segregation, wild-type cells were transformed with a plasmid copy of psaC, containing an insertion of the kan^r gene, increasing the size of the EcoRI fragment from 8.0 kb (wild type, wt) to 9.4 kb. Transformants were grown under photoautotrophic or light-activated heterotrophic conditions; data of the sixth and the third passage, respectively, are presented. (D) In the presence of pRL1342-encoded SynToc75 and selection with erythro- mycin and kanamycin, the chromosomal wild-type copies of synToc75 disappeared, as indicated by the absence of the 4.6-kb EcoRI fragment (synToc75-kan^r) and also the 10.5-kb fragment (ΔsynToc75-kan^rORF). The control (synToc75-kan^r) shows the presence of chromosomal wild-type and mutant synToc75. Only data for photoautotrophically grown Synechocystis transformants are presented.

Figure 4

Multiple sequence alignments of SynToc75 with related proteins. (A) Summary of sequence alignments of pea Toc75, the D15 homologs, and D15-related proteins with SynToc75. The gray shaded areas represent the regions of sequence similarity with SynToc75, as indicated by blast 2.0 (pea Toc75, D15, and D15-related proteins) or psi-blast (HecB of Erwinia chrysanthemi, ShlB of Serratia marcescens). The black boxes indicate the location of the C-terminal motif. The C-terminal motifs of the putative (put.) OMP of Treponema pallidum and ShlB are less conserved, as indicated by a different pattern. (B) Multiple sequence alignment of the C-terminal regions of SynToc75 and the D15-related proteins. The black boxes indicate high sequence similarity (>90%), the gray shading indicates low sequence similarity (>40%) (31).