The Role of Slr0151, a Tetratricopeptide Repeat Protein from Synechocystis sp. PCC 6803, during Photosystem II Assembly and Repair

Abstract

The assembly and repair of photosystem II (PSII) is facilitated by a variety of assembly factors. Among those, the tetratricopeptide repeat (TPR) protein Slr0151 from Synechocystis sp. PCC 6803 (hereafter Synechocystis) has previously been assigned a repair function under high light conditions (Yang et al., 2014). Here, we show that inactivation of slr0151 affects thylakoid membrane ultrastructure even under normal light conditions. Moreover, the level and localization of Slr0151 are affected in a variety of PSII-related mutants. In particular, the data suggest a close functional relationship between Slr0151 and Sll0933, which interacts with Ycf48 during PSII assembly and is homologous to PAM68 in Arabidopsis thaliana. Immunofluorescence analysis revealed a punctate distribution of Slr0151 within several different membrane types in Synechocystis cells.

Keywords: Synechocystis; TPR protein; biogenesis center; photosystem II; thylakoid membrane.

FIGURE 1

Steady-state levels of the indicated PSII-related proteins in the slr0151^- mutant (A) and of Slr0151 in the indicated PSII mutants (B). Each value is expressed relative to that in the wild type. Total cell proteins were isolated, fractionated by SDS-PAGE, blotted onto nitrocellulose membranes and detected immunologically. Signals were quantified with AIDA software (version 3.52.046) after densitometrical scanning. Values plotted are means ± SD of at least three independent experiments. Significance according to Student’s t-test with an error probability of 5 and 1% is indicated by one and two asterisks, respectively.

FIGURE 2

Transmission electron microscopy of wild-type Synechocystis(A) and slr0151^- mutant cells (B–D). Different stages during the cell division of the slr0151^- strain are shown; early stage of the cell division (B), late stage of cell division right before cytokinesis (C) and non-dividing single cell (D). Intracellular components are labeled: Outer membrane (white arrowhead), plasma membrane (black arrowhead), thylakoids (white asterisks) and carboxysome (c). Bars = 500 nm.

FIGURE 3

Membrane localization of Slr0151. Sucrose step-gradient centrifugation was used to separate membrane types from wild-type Synechocystis cells. Fraction II corresponds to the plasma membrane (PM) and fraction V contains pratA-defined biogenic membranes (PDMs) + thylakoid membrane (TMs). For fractions I–IV 10% of the total volume of each fraction was loaded, while only 0.2% of fraction V was analyzed (Schottkowski et al., 2009a).

FIGURE 4

Distribution of Slr0151 between PDMs and TMs. (A) Wild-type fraction V (see Figure 3) was centrifuged on linear sucrose gradients in order to separate wild-type PDMs from TMs. (B) Distribution of Slr0151 between PDMs and TMs in the indicated PSII mutants. (C) Distribution of various PSII-related proteins in slr0151^- cells. The part of the gradient from 20 to 60% sucrose was apportioned into 14 fractions, which were analyzed by immunoblotting using the antibodies indicated on the right. Fractions 1–6 represent PDMs, and fractions 7–14 represent TMs. To facilitate comparison between gradients, sample volumes were normalized to the volume of fraction 7 that contained 40 μg of protein.

FIGURE 5

Subcellular localization of Slr0151 by immunofluorescence analysis of wild-type Synechocystis cells. Cells grown under normal light conditions (A) and cells that had been exposed to high light for 1 h (B) were treated with αSlr0151 antibody and detected with an Alexa-488-coupled secondary antibody. The constituent subpanels show Z-montages (200 nm spacing) of each separate channel as well as the merged channel. The line plots of relative fluorescence signals are derived from scans of the area defined by the white circle around the indicated cells, and correspond to the Z-slice number of the analyzed slice (given at the top). (C) For controls, cells were grown under normal conditions. In the upper row, Synechocystis wild-type cells were immunostained with αRbcL and visualized with Alexa-488 coupled secondary antibody. In the middle row, slr0151^- mutant cells have been treated with the αSlr0151 antibody followed by Alexa-488-coupled secondary antibody. In the bottom row, wild-type cells were probed with Alexa-488-coupled secondary antibody alone. AF, autofluorescence of chlorophyll; scale bar 1 μm.