The C terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane

Abstract

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.

FIGURE 1.

The Alb3 C-terminal domain interacts with cpSRP43. A, membrane topology of Alb3. Mature Alb3 is a polytopic membrane protein comprising five TMs. The topology prediction was generated by the use of the TopPred II software (57). The luminal, stromal, and C-terminal (C) domains are labeled. N, N terminus; B, analysis of cpSRP43-A3CT complex formation. cpSRP43 alone (black line) or a mixture of cpSRP43 and A3CT in a 1:2 ratio (gray line) was subjected to SEC. Peak fractions were analyzed by SDS-PAGE (inset). The standard proteins used for calibration are indicated by black triangles: γ-globulin (158 kDa), bovine serum albumin (66 kDa), ovalbumin (44 kDa), carboanhydrase (29 kDa), myoglobin (17 kDa), and cytochrome c (12.4 kDa). C, determination of binding affinities for cpSRP43-A3CT interaction ITC. Analysis of the titration isotherms resulted in a K_d of 9.7 μm for A3CT and a stoichiometry of 1:1. Data analysis was performed using the Origin 7.0 software. See also Table 1.

FIGURE 2.

A3CT is intrinsically disordered and folds upon binding to cpSRP43. A, analysis of A3CT using SEC. A3CT (molecular mass 15 kDa) shows an aberrant migration behavior corresponding to an apparent molecular mass of 38 kDa. The protein standards (black triangles) are the same as in Fig. 1B. mAU, milliabsorbance units; B, sequence analysis predicts A3CT as intrinsically disordered. PONDR (28) analysis of Alb3 (1–462) from A. thaliana. A large segment of predicted disorder is indicated in the C-terminal region. mdeg, millidegrees. C, analysis of A3CT secondary structure using CD spectroscopy indicates that A3CT is unfolded in solution. Far-UV CD spectra of A3CT (blue line), cpSRP43 (black line), and A3CTFL cpSRP43 in an equimolar mixture (red line) are shown. D, A3CT folds and adopts an α-helical conformation upon binding to cpSRP43. The difference spectrum obtained by subtracting the CD spectrum of cpSRP43 (black line in C) from the spectrum of cpSRP43 and A3CT mixture (red line in C) shows two minima at around 207 and 222 nm typical for α-helices.

FIGURE 3.

Alb3-cpSRP43 interaction requires two positively charged motifs absent in Alb4. A, multiple sequence alignment of Alb3 C-terminal domains from different plants. Four conserved positively charged motifs (motif I–IV) are present. Sequence numbers for A. thaliana are given above the sequence. Species names are given on the left. Highly conserved residues are shown in dark blue, less conserved residues are in light blue. P. sativum, Pisum sativum; V. vinifera, Vitus vinifera; P. trifoliata, Ptelea trifoliata; O. sativa, Oryza sativa. B, schematic representation of the A3CT constructs used in this study. Dark gray areas indicate the conserved motifs I–IV that have been identified in A. The relative affinities of the truncated A3CT constructs for cpSRP43 as compared with A3CT (containing motifs I–IV) derived from ITC are indicated on the right (see Table 1). For details of the A3CT variants, see the supplemental material. C, sequence alignment of Alb3 and Alb4 C-terminal domains from A. thaliana. The conserved motifs I and III identified in Alb3 proteins in A are present in Alb4 but modified (indicated by an asterisk); motifs II and IV are absent.

FIGURE 4.

Alb3 interaction requires cpSRP43 chromodomains 2 and 3. A, schematic representation of cpSRP43 constructs used in this study. The domain organization of cpSRP43 with chromodomains (CD1–3) and ankyrin repeats (Ank1–4) is shown. The relative affinities (rel. aff.) of the truncated cpSRP43 constructs for A3CT as compared with cpSRP43 (derived from ITC, Table 1 and supplemental Fig. S4) are indicated on the right. B, sequence alignment of the three chromodomains of cpSRP43 (A. thaliana) (CD1–3) as compared with canonical chromodomains from HP1 and polycomb (Drosophila melanogaster). The positions of the three aromatic residues forming the aromatic cage in HP1 and PC are indicated by black dots. The positions of the three point mutations (Y269A/W291A/D293A) in cpSRP43 CD2 used in interaction studies are indicated by black stars. Highly conserved residues are shown in dark blue, and less conserved residues are in light blue. C, ribbon representation of the structure of the HP1 in complex with the histone H3 tail (PDB (Protein Data Bank) code: 1kne). HP1 is shown in blue, and the peptide of the histone H3 tail is represented in orange. Canonical chromodomains bind methylated lysines (M3K) in a conserved aromatic cage. The adjacent peptide extends the β-sheet of the chromodomain. Panels C and D were created using PyMOL (58). D, ribbon representation of cpSRP43 CD2 (model based on the crystal structure of CD1, PDB code 3deo; in green). The third aromatic residue forming the aromatic cage is absent in cpSRP43 chromodomains, and the aromatic cage is not assembled in the absence of an interacting peptide. The arrow indicates a possible movement of the N-terminal region upon binding of a histone-like peptide derived from A3CT.

FIGURE 5.

cpSRP43 can interact with Alb3 and cpSRP54 simultaneously. A, analysis of complex formation using a competition pulldown assay. cpSRP43-A3CT-His₆ (labeled 1 and 3) was incubated with increasing amounts of cpSRP54 M-domain (labeled 2) up to 12.5 molar excess (for details, see the supplemental material). A3CT, cpSRP43, and the cpSRP54 M-domain form a trimeric complex. B, thermodynamic cycle of trimeric complex formation by cpSRP43 (labeled 43), A3CT, and the RRKR peptide (RRKR, representing the M-domain of cpSRP54). The two binding events, A3CT and RRKR peptide binding to cpSRP43, are coupled and show negative cooperativity. The affinities were derived from the ITC data (Table 1).

FIGURE 6.

Schematic view on Alb3 function in post-translational targeting. cpSRP54 and cpSRP43 form cpSRP, which sequesters LHCPs into a soluble transit complex (LHCP-cpSRP43-cpSRP54) (step 1). The Alb3 C-terminal domain contains four motifs enriched in positively charged residues (motifs I–IV) (step 2) and recruits the transit complex to the membrane utilizing motifs II and IV. A3CT folds upon binding to cpSRP43 (step 3). cpSRP54 and A3CT can bind simultaneously to CD2. Thereby the transit complex is recruited to the membrane, and cpSRP54 can interact with the membrane-bound receptor cpFtsY. The C-terminal domain of Alb4 lacks motifs II and IV and therefore does not bind cpSRP43 (step 4).