The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in‐solution and in‐gel digestion

From individual localization and large‐scale proteomic studies, we know that stroma‐exposed thylakoid membranes harbor part of the machinery performing the light‐dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in‐solution and in‐gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.g. transporters, in stroma thylakoids prepared from Arabidopsis thaliana (L.) Heynh Columbia‐0 leaves. In‐solution digestion with chymotrypsin yielded the largest number of peptides, but in combination with methanol extraction resulted in identification of the largest number of membrane proteins. Although less efficient in extracting peptides, in‐gel digestion with trypsin and chymotrypsin led to identification of additional proteins. We identified a total of 58 proteins including 44 membrane proteins. Almost half are known thylakoid proteins with roles in photosynthetic light reactions, proteolysis and import. The other half, including many transporters, are not known as chloroplast proteins, because they have been either curated (manually assigned) to other cellular compartments or not curated at all at the plastid protein databases. Transporters include ATP‐binding cassette (ABC) proteins, transporters for K⁺ and other cations. Other proteins either have a role in processes probably linked to photosynthesis, namely translation, metabolism, stress and signaling or are contaminants. Our results indicate that all these proteins are present in stroma thylakoids; however, individual studies are required to validate their location and putative roles. This study also provides strategies complementary to traditional methods for identification of membrane proteins from other cellular compartments.