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The vesicle-inducing protein in plastids 1 (VIPP1) is crucial for the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. Although VIPP1 has been studied for many years, it is not well understood how this protein interacts with target membranes. Chlamydomonas encodes two VIPP paralogs. Here, we investigated the effect of total VIPP loss in Chlamydomonas. Using CRISPR/Cas9, the endogenous VIPP genes were knocked out and replaced by a VIPP1 gene under control of a riboswitch and vitamin-repressible promotor. Induced loss of VIPP1 in these lines resulted in a chlorotic phenotype under non-stressed conditions in combination with an upregulated cpUPR, loss of photosystems as well as induction of autophagy. In a second approach, VIPP1s ability to interact with membranes via its N-terminal amphipathic alpha helical
(AHa) domain was disturbed by introducing mutations altering the hydrophobic face. Therefore, a VIPP1 knockout mutant was generated via CRISPR/Cas9 and supertransformed with VIPP1-mNeonGreen genes encoding the variants with mutations in the AHa domain. Reduced hydrophobicity of the AHa domain led to a similar phenotype as the VIPP1 knockout hinting to the crucial character of the domain for membrane interaction. Our data point to an important role of the AHa domain of VIPP1 for recognizing and stabilizing stressed chloroplast membranes in Chlamydomonas.
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