A mutant small heat shock protein with increased thylakoid association provides an elevated resistance against UV-B damage in synechocystis 6803

Abstract

Besides acting as molecular chaperones, the amphitropic small heat shock proteins (sHsps) are suggested to play an additional role in membrane quality control. We investigated sHsp membrane function in the model cyanobacterium Synechocystis sp. PPC 6803 using mutants of the single sHsp from this organism, Hsp17. We examined mutants in the N-terminal arm, L9P and Q16R, for altered interaction with thylakoid and lipid membranes and examined the effects of these mutations on thylakoid functions. These mutants are unusual in that they retain their oligomeric state and chaperone activity in vitro but fail to confer thermotolerance in vivo. We found that both mutant proteins had dramatically altered membrane/lipid interaction properties. Whereas L9P showed strongly reduced binding to thylakoid and model membranes, Q16R was almost exclusively membrane-associated, properties that may be the cause of reduced heat tolerance of cells carrying these mutations. Among the lipid classes tested, Q16R displayed the highest interaction with negatively charged SQDG. In Q16R cells a specific alteration of the thylakoid-embedded Photosystem II (PSII) complex was observed. Namely, the binding of plastoquinone and quinone analogue acceptors to the Q(B) site was modified. In addition, the presence of Q16R dramatically reduced UV-B damage of PSII activity because of enhanced PSII repair. We suggest these effects occur at least partly because of increased interaction of Q16R with SQDG in the PSII complex. Our findings further support the model that membrane association is a functional property of sHsps and suggest sHsps as a possible biotechnological tool to enhance UV protection of photosynthetic organisms.

FIGURE 1.

Subcellular distribution of WT and mutant Hsp17 proteins in heat acclimated cells. Cells grown photoautrophically at 30 °C were exposed to 42 °C for 3 h, and then cytosolic (soluble) and thylakoid (pellet) fractions were isolated and subjected to Western analysis with anti-Hsp17 antibody.

FIGURE 2.

Binding of Hsp17 proteins to liposomes made of total polar lipids isolated from heat-treated cells. A, purity of recombinant proteins was assayed by SDS-PAGE and revealed by Coomassie blue staining. Low molecular weight markers (LMW) display protein bands referring to molecular masses of 94, 67, 43, 30, 20, and 14 kDa, respectively. B, purified proteins were incubated with liposomes for 1 h and then fractionated into lipid bound (pellet) and soluble (supernatant) fractions. The samples were solubilized under reducing conditions and analyzed by SDS-PAGE and Coomassie blue staining. C, the binding assay was repeated with vesicles made of ¹⁴C-labeled lipids. The arrow indicates the band corresponding to the Hsp17 associated with ¹⁴C-labeled lipids. Lipid content of the pelleted Q16R Hsp17 fraction solubilized under reducing conditions was revealed by fluorography.

FIGURE 3.

Interaction of WT and mutant Hsp17 proteins with monomolecular lipid layers of total polar lipids (A) and SQDG (B) isolated from heat-treated cells. A, increasing concentrations of Hsp17 proteins (WT, Q16R, and L9P as circles, triangles, and diamonds, respectively) were injected underneath lipid layers, and equilibrium surface pressures were recorded. B, 4.2 μg/ml of each sHsp (WT, Q16R, and L9P as solid, dashed, and dotted lines, respectively) was injected into the buffer beneath the lipid layer, and development of protein-lipid binding was monitored by following the surface pressure increase.

FIGURE 4.

Relaxation of flash induced chlorophyll fluorescence in heat preconditioned (42 °C, 3 h) WT (circles) and Q16R (triangles) cells. Fluorescence excitation was achieved with a single turnover saturating flash at t = 1 ms. Subsequent fluorescence relaxation was measured in the absence of electron transport inhibitors (A) and in the presence of 10 μm (3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) (B). The curves shown were normalized to the same initial amplitude and corrected according to Joliot and Joliot (24) to consider nonlinear relationships of fluorescence yield and Q^–_A concentration.

FIGURE 5.

PSII activity measured as the rate of oxygen evolution in the WT (white bars) and Q16R mutant (striped bars) cells before (A) and after heat treatment (42 °C, 3 h) (B). The activity was tested in the presence of different electron acceptors such as 2,5-DMBQ, 2,5-DCBQ, and pBQ (0.5 mm each). The error bars represent standard errors obtained from three repetitions.

FIGURE 6.

Effect of Hsp17 mutations on oxygen evolving activity. Heat-preconditioned (42 °C, 3 h) WT (circles), Q16R (triangles), and L9P (diamonds) strains were exposed to UV-B radiation for 2 h followed by a recovery period in visible light (VIS) for 1 h. Oxygen evolving activities were measured at the indicated time points. The results are expressed as percentages of the oxygen evolution rate measured at time 0. The error bars represent standard errors obtained from three repetitions. The experiments were performed in the absence (A) or presence (B) of the protein synthesis inhibitor lincomycin.

FIGURE 7.

Change in the total D1 protein content in heat preconditioned WT (circles) and Q16R (triangles) cells challenged by UV-B stress. D1 content in samples was followed during UV-B stress and subsequent recovery (A) or tested upon prolonged UV-B treatment in the presence of lincomycin (B). The data are obtained by densitometry of the Western blots and are the means of three parallel experiments. The results are expressed as percentages of the initial D1 protein level. VIS, visible light.