Introduction
Intrinsic membrane proteins (IMPs) represent an extremely diversified group that represents roughly 30 % of the cellular proteome (Wallin and Von Heijne, 1998). Tail anchored (TA) proteins share a simple type II topology with a cytosolically exposed N-terminus and a single C-terminal transmembrane domain (TMD) within the last 50 amino acids (AAs) of the polypeptide stretch. Even though they constitute 3-5% of eukaryotic IMPs (Kalbfleisch et al., 2007), TA proteins fulfill a multitude of essential biological functions like apoptosis (Jiang et al., 2014), vesicle transport (van Berkel et al., 2020) and organelle biogenesis (Sommer et al., 2013). The guided entry of TA proteins (GET) pathway facilitates the post-translational targeting of this subset of IMPs. After translation is terminated, the pretargeting complex captures the TA protein and hands it over to the central ATPase of the GET pathway, Get3 (Wang et al., 2010). Thereafter the TA protein is passed on to its cognate receptor, which functions as an insertase (Wang et al., 2014). For reasons of simplicity, we will generally refer to “Get” factors and plant specific elements will be denoted in all capital letters, in agreement with Arabidopsis thaliana nomenclature.
A characteristic trait of Get3 is the formation of a symmetric homodimer equipped with a static ATPase domain and a flexible α-helical domain that is sensitive to nucleotide binding. This nucleotide binding state induces the formation of a hydrophobic groove or chamber capable of binding TA substrate (Mateja et al., 2009; Bozkurt et al., 2009, Mateja et., 2015, Barlow et al., 2023). In a cellular context, Get3 also has a secondary function in eukaryotes by acting as an ATP-independent holdase under energy limiting conditions and oxidative stress (Powis et al., 2013; Voth et al., 2014). Get3 and its orthologues TRC40/ArsA are highly conserved in all domains of life and have diversified into three clades, namely clade a, clade bc and clade d (Xing et al., 2017; Barlow et al., 2023). In contrast to metazoans and fungi, which solely contain clade a Get3 homologues, photosynthetic organisms contain multiple Get3 homologues from all clades (Xing et al., 2017; Bodensohn et al., 2019; Barlow et al., 2023). The three clades differ in their amion acid (AA) composition, which results in different domain architectures. In the majority of the cases clade a proteins have a cytosolic and clade bc an organellar localization (Xing et al., 2017; Bodensohn et al., 2019). Depending on the prokaryotic or eukaryotic origin of a photosynthetic organism, clade d proteins have a cytosolic or organellar localization, respectively (Barlow et al., 2023).
Arabidopsis thaliana has four Get3 homologues, namelyAt GET3A, At GET3B, At GET3C and At GET3D localized to cytosol, chloroplasts, mitochondria and chloroplasts, respectively (Duncan et al., 2013; Xing et al., 2017; Bodensohn et al., 2019; Barlow et al., 2023). Loss of function mutations in any of the clade a or clade bc orthologues renders the resulting plant largely indistinguishable from the wild-type (Xing et al., 2017). This is also the case in yeast and nematodes in which the deletion of Get3 leads to conditional growth defects (Schuldiner et al., 2008; Tseng et al., 2007). In mammals however, the disruption of TRC40 is embryo lethal (Mukhopadhyay et al., 2006).
In photosynthetic eukaryotes, plastids constitute one of the most complex systems in terms of protein sorting. 95 % of their proteome is encoded by nuclear genes and precursor proteins are synthesized on cytosolic ribosomes to be imported post-translationally. This is facilitated by two multimeric translocons embedded in the outer (TOC) and inner (TIC) envelope that form a continuum from the cytosol, through the intermembrane space (IMS) into the stroma (Liu et al., 2023). Once arrived in the stroma, precursor proteins can be directed to numerous translocation machineries imbedded within the inner envelope or thylakoid membrane. This leads to six different sub organellar localizations that each can individually be accessed with different sets of protein targeting networks (Jarvis and López-Juez, 2013). Taking the pivotal role of plastids into account, the targeting and translocation networks must operate with high fidelity to ensure proper organellar biogenesis, repair and functionality. This is reflected by the deficiencies in plant fitness that arise when components of these are disrupted (Bauer et al., 2000; Amin et al., 1999; Pilgrim et al., 1998; Sundberg et al., 1997).
One intensively studied stromal targeting pathway is the post-translational cpSRP pathway, which targets a single population of IMPs, namely light harvesting chlorophyll binding proteins, to the thylakoid membrane (Schuenemann et al., 1998; Ziehe et al., 2018). CpSRP is composed of cpSRP54 (henceforth referred to as SRP54), the plastidic homologue of a highly conserved subunit of the cytosolic SRP, and a plant specific targeting factor, cpSRP43 (SRP43 from here on). SRP54 can also interact with the plastidic 70S ribosome and mediates co-translational transport of substrate proteins to its respective insertase, ALB3 and the SecYE translocon (Hristou et al., 2019; Moore et al., 2003).
The terminal insertase ALB3 belongs to the Oxa1 superfamily of membrane protein biogenesis factors which also includes the ER resident Get1 (Anghel et al., 2017). In plants, an additional homologue of ALB3 exists, ALB4 (Gerdes et al., 2006). ALB4 is involved in the assembly and stabilization of ATP synthase intermediates (Benz et al., 2009). Furthermore, subsequent analyses displayed that ALB4 works at least partially together with ALB3 and participates in the biogenesis of a subset of thylakoid membrane proteins. (Trösch et al., 2015).
ALB4 also interacts with STIC2, a protein identified in a genetic screen for the suppressors of chlorotic tic40 , an A. thaliana mutant lacking the chloroplast inner envelope protein TIC40. The STIC system is described as a two component network composed of STIC1 (ALB4) and STIC2, which acts together in a shared pathway with cpSRP54 and cpFtsY (Bédard et al., 2017). STIC2 shares homology with the bacterial YbaB,(Link et al., 2008). Recently, STIC2, was identified as a factor associated with plastidic ribosomes translating the photosystem (PS) II reaction center protein D1, is likely involved in co-translational sorting of other plastid-encoded multi-pass IMPs (Stolle et al., 2024).
In a previous study, we provided evidence that the At GET3B can selectively bind SECE1 and shows a genetic interaction with components of the cpSRP system. Furthermore, mutant plants showed altered levels of photosynthesis-related proteins (Anderson et al., 2021). This prompted us to investigate if GET3B interacts with constituents of the cpSRP and STIC systems, with an emphasis on their cognate receptors ALB3 and ALB4, respectively. We hypothesized that GET3B might play a crucial role in the targeting and membrane integration of specific proteins within chloroplasts, thereby contributing to the biogenesis and functionality of chloroplast membranes. To address this hypothesis, we employed a combination of biochemical, molecular, fluorometric and genetic approaches to provide evidence that GET3B physically interacts with the C-terminus of ALB3 as well as ALB4 and genetically interacts with components of the STIC and cpSRP pathways, and that its disruption has an effect on chloroplast biogenesis and function.