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.