cpSRP43 is a novel chaperone specific for light-harvesting chlorophyll a,b-binding proteins

Abstract

The biosynthesis of most membrane proteins is directly coupled to membrane insertion, and therefore, molecular chaperones are not required. The light-harvesting chlorophyll a,b-binding proteins (LHCPs) present a prominent exception as they are synthesized in the cytoplasm, and after import into the chloroplast, they are targeted and inserted into the thylakoid membrane. Upon arrival in the stroma, LHCPs form a soluble transit complex with the chloroplast signal recognition particle (cpSRP) consisting of an SRP54 homolog and the unique cpSRP43 composed of three chromodomains and four ankyrin repeats. Here we describe that cpSRP43 alone prevents aggregation of LHCP by formation of a complex with nanomolar affinity, whereas cpSRP54 is not required for this chaperone activity. Other stromal chaperones like trigger factor cannot replace cpSRP43, which implies that LHCPs require a specific chaperone. Although cpSRP43 does not have an ATPase activity, it can dissolve aggregates of LHCPs similar to chaperones of the Hsp104/ClpB family. We show that the LHCP-cpSRP43 interaction is predominantly hydrophobic but strictly depends on an intact DPLG motif between the second and third transmembrane region. The cpSRP43 ankyrin repeats that provide the binding site for the DPLG motif are sufficient for the chaperone function, whereas the chromodomains are dispensable. Taken together, we define cpSRP43 as a highly specific chaperone for LHCPs in addition to its established function as a targeting factor for this family of membrane proteins.

FIGURE 1.

cpSRP43 is an LHCP-specific chaperone. A, schematic representation of cpSRP43 variants used in this study. The domain organization of cpSRP43 with chromodomains (CD1–3) (light gray ovals) and ankyrin repeats (Ank1–4) (dark gray rectangles) is shown. B, cpSRP43 alone is able to keep LHCP in solution. Denaturated LHCP (8 m urea) was diluted 40-fold in the absence or presence of the proteins indicated above each lane or in the presence of 0.5% SDS and divided into supernatant and pellet by centrifugation followed by SDS-PAGE/CBB staining analysis. Only the supernatant fraction is depicted. Bands are labeled on the right side of the gel as follows: cpSRP54 with 54, cpSRP43 with 43, and the C-terminal domain of Alb3 with A3CT. The three characteristic bands for LHCP representing monomer, dimer, and higher oligomers are marked in the gel with an asterisk. C, the LHCP-cpSRP43 interaction is predominantly hydrophobic. LHCP and cpSRP43 form a stable, salt-resistant complex. His-tagged LHCP (marked with an asterisk) was refolded in the presence of non-tagged cpSRP43 (labeled 43). Complex formation was analyzed by pulldown experiments under low salt (150 mm NaCl) or high salt (1 m NaCl) conditions. No binding of cpSRP43 was observed in the absence of LHCP (Control). D, cpSRP43 but not trigger factor prevents the aggregation of LHCP. Aggregation of LHCP is indicated by the increase in right angle light scattering at 650 nm after 100-fold dilution of denaturated LHCP (final concentration 0.12 μm) and was monitored in the absence (blue line) or presence of 0.13 μm cpSRP43 (red line) or 0.13 μm trigger factor (black line). The inset shows the analysis of cpSRP43 (lane 1) and the cpSRP43-LHCP complex (lane 2) by clear native PAGE followed by CBB staining. Formation of the cpSRP43-LHCP complex is indicated by a band shift to lower mobility. E, solubilization of aggregated LHCP and LHCP-L201K mutant was monitored in the absence or presence of cpSRP43. The turbidity of aggregated LHCP or LHCP-L201K was set to 100%, and disaggregation was monitored by the decrease in right angle light scattering at 650 nm. Blue line and red line, LHCP in the absence (blue line) or presence (red line) of cpSRP43. Black line and green line, LHCP-L201K in the absence (black line) or presence of cpSRP43 (green line). Only in the presence of cpSRP43 does disaggregation occur. F, the cpSRP43 ankyrin repeats carry the chaperone function. Denaturated LHCP (8 m urea) was diluted 40-fold in the absence or presence of the different cpSRP43 variants (indicated above each lane) or in the presence of 0.5% SDS. The samples were divided into supernatant and pellet by centrifugation followed by SDS-PAGE/CBB staining. Only the supernatant fraction is depicted. Bands are labeled on the right using the abbreviations given in panel A. The three characteristic bands for LHCP representing monomer, dimer, and higher oligomers are marked with an asterisk.

FIGURE 2.

The LHCP L18 region is essential for interaction with cpSRP43. A, schematic representation of the LHCP topology with three transmembrane helices (TM1–3). The sequence of the L18 peptide is given from the major LHCP (Lhcb1 P. sativum), and the DPLG motif is highlighted in bold. The residue numbers for the truncation constructs used are indicated by an arrow. B and C, the L18 region is strictly required for cpSRP43-LHCP complex formation. Denatured LHCP and LHCP-L201K (B) and LHCPΔTM3 or LHCPΔL18ΔTM3 (C) were diluted 40-fold into buffer in the presence or absence of 0.5% SDS or cpSRP43. The samples are divided into supernatant and pellet by centrifugation followed by SDS-PAGE/CBB staining. Only the supernatant fraction is depicted. Bands corresponding to LHCP variants are labeled with an asterisk, and the band corresponding to cpSRP43 is labeled on the right with 43. Note: The dimer of LHCPΔTM3 comigrates with cpSRP43. D and E, L18 competes with LHCP for binding to cpSRP43. Formation of the cpSRP43-LHCP complex (D) and the cpSRP43-LHCPΔTM3 (E) complex was analyzed in the presence of increasing amounts of L18 peptide (0–500 μm). The samples were prepared as in panel B. Bands are labeled as follows: cpSRP43 with 43, LHCP with LHCP, LHCPΔTM3 with ΔTM3. Note: In E, the dimer of LHCPΔTM3 comigrates with cpSRP43. For quantification of bound LHCP, the lowest band representing LHCP monomer was analyzed by ImageJ (54), and the band intensity was plotted against L18 concentration.