Distinct Albino3-dependent and -independent pathways for thylakoid membrane protein insertion

The homologous proteins Oxa1, YidC, and Alb3 mediate the insertion of membrane proteins in mitochondria, bacteria, and chloroplast thylakoids, respectively. Depletion of YidC in Escherichia coli affects the integration of every membrane protein studied, and Alb3 has been shown previously to be required for the insertion of a signal recognition particle (SRP)-dependent protein, Lhcb1, in thylakoids. In this study we have analyzed the "global" role of Alb3 in the insertion of thylakoid membrane proteins. We show that insertion of two chlorophyll-binding proteins, Lhcb4.1 and Lhcb5, is almost totally blocked by preincubation of thylakoids with anti-Alb3 antibodies, indicating a requirement for Alb3 in the insertion pathway. Insertion of the related PsbS protein, on the other hand, is unaffected by Alb3 antibodies, and insertion of a group of SRP-independent, signal peptide-bearing proteins, PsbX, PsbW, and PsbY, is likewise completely unaffected. Proteinase K is furthermore able to completely degrade Alb3, but this treatment does not affect the insertion of these proteins. Among the thylakoid proteins studied here, Alb3 requirement correlates strictly with a requirement for stromal factors and nucleoside triphosphates. However, the majority of proteins tested do not require Alb3 or any other known form of translocation apparatus.     

Chloroplast SecE: evidence for spontaneous insertion into the thylakoid membrane

SecE, an essential component of the bacterial SecAYEG translocase, mediates protein translocation across the cytoplasmic membrane. In the thylakoid membranes of chloroplasts an SecE homologue, cpSecE, has recently been identified. In this report we show that insertion of cpSecE does not require stromal extract, indicating that signal recognition particle is not involved. Removal of nucleoside triphosphates has apparently no effect on the integration, again ruling out an involvement of SRP or its partner protein, FtsY. The use of well-known inhibitors of the Sec- and Tat pathways, sodium azide and nigericin, respectively, also had no influence on membrane insertion. The data presented here point towards cpSecE as another passenger of a wholly spontaneous import/insertion pathway in the thylakoids of chloroplasts.     

A homolog of Albino3/OxaI is essential for thylakoid biogenesis in the cyanobacterium Synechocystis sp. PCC6803

YidC/OxaI play essential roles in the insertion of a wide range of membrane proteins in Eschericha coli and mitochondria, respectively. In contrast, the chloroplast thylakoid homolog Albino3 (Alb3) facilitates the insertion of only a specialized subset of proteins, and the vast majority insert into thylakoids by a pathway that is so far unique to chloroplasts. In this study, we have analyzed the role of Alb3 in the cyanobacterium Synechocystis sp. PCC6803, which contains internal thylakoids that are similar in some respects to those of chloroplasts. The single alb3 gene (slr1471) was disrupted by the introduction of an antibiotic cassette, and photoautotrophic growth resulted in the generation of a merodiploid species (but not full segregation), indicating an essential role for Alb3 in maintaining the photosynthetic apparatus. Thylakoid organization is lost under these conditions, and the levels of photosynthetic pigments fall to approximately 40% of wild-type levels. Photosynthetic electron transport and oxygen evolution are reduced by a similar extent. Growth on glucose relieves the selective pressure to maintain photosynthetic competence, and under these conditions, the cells become completely bleached, again indicating that Alb3 is essential for thylakoid biogenesis. Full segregation could not be achieved under any growth regime, strongly suggesting that the slr1471 open reading frame is essential for cell viability.     

Chloroplast YidC homolog Albino3 can functionally complement the bacterial YidC depletion strain and promote membrane insertion of both bacterial and chloroplast thylakoid proteins

A new component of the bacterial translocation machinery, YidC, has been identified that specializes in the integration of membrane proteins. YidC is homologous to the mitochondrial Oxa1p and the chloroplast Alb3, which functions in a novel pathway for the insertion of membrane proteins from the mitochondrial matrix and chloroplast stroma, respectively. We find that Alb3 can functionally complement the Escherichia coli YidC depletion strain and promote the membrane insertion of the M13 procoat and leader peptidase that were previously shown to depend on the bacterial YidC for membrane translocation. In addition, the chloroplast Alb3 that is expressed in bacteria is essential for the insertion of chloroplast cpSecE protein into the bacterial inner membrane. Surprisingly, Alb3 is not required for the insertion of cpSecE into the thylakoid membrane. These results underscore the importance of Oxa1p homologs for membrane protein insertion in bacteria and demonstrate that the requirement for Oxa1p homologs is different in the bacterial and thylakoid membrane systems.     

A second thylakoid membrane-localized Alb3/OxaI/YidC homologue is involved in proper chloroplast biogenesis in Arabidopsis thaliana

The integral membrane proteins Alb3, OxaI, and YidC belong to an evolutionary conserved protein family mediating protein insertion into the thylakoid membrane of chloroplasts, the inner membrane of mitochondria, and bacteria, respectively. Whereas OxaI and YidC are involved in the insertion of a wide range of membrane proteins, the function of Alb3 seems to be limited to the insertion of a subset of the light-harvesting chlorophyll-binding proteins. In this study, we identified a second chloroplast homologue of the Alb3/OxaI/YidC family, named Alb4. Alb4 is almost identical to the Alb3/OxaI/YidC domain of the previously described 110-kDa inner envelope protein Artemis. We show that Alb4 is expressed as a separate 55-kDa protein and that Artemis was identified mistakenly. Alb4 is located in the thylakoid membrane of Arabidopsis thaliana chloroplasts. Analysis of an Arabidopsis mutant (Salk_136199) and RNA interference lines with a reduced level of Alb4 revealed chloroplasts with an altered ultrastructure. Mutant plastids are larger and more spherical in appearance, and the grana stacks within the mutant lines are less appressed than in the wild-type chloroplasts. These data indicate that Alb4 is required for proper chloroplast biogenesis.     

Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.     

YidC/Alb3/Oxa1 Family of Insertases

The YidC/Alb3/Oxa1 family functions in the insertion and folding of proteins in the bacterial cytoplasmic membrane, the chloroplast thylakoid membrane, and the mitochondrial inner membrane. All members share a conserved region composed of five transmembrane regions. These proteins mediate membrane insertion of an assorted group of proteins, ranging from respiratory subunits in the mitochondria and light-harvesting chlorophyll-binding proteins in chloroplasts to ATP synthase subunits in bacteria. This review discusses the YidC/Alb3/Oxa1 protein family as well as their function in membrane insertion and two new structures of the bacterial YidC, which suggest a mechanism for membrane insertion by this family of insertases.     

The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.     

Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts

Members of the YidC/Oxa1/Alb3 protein family facilitate the insertion, folding and assembly of proteins of the inner membranes of bacteria and mitochondria and the thylakoid membrane of plastids. All homologs share a conserved hydrophobic core region comprising five transmembrane domains. On the basis of phylogenetic analyses, six subgroups of the family can be distinguished which presumably arose from three independent gene duplications followed by functional specialization. During evolution of bacteria, mitochondria and chloroplasts, subgroup-specific regions were added to the core domain to facilitate the association with ribosomes or other components contributing to the substrate spectrum of YidC/Oxa1/Alb3 proteins.     

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.     

The evolution of YidC/Oxa/Alb3 family in the three domains of life: a phylogenomic analysis

Background  

YidC/Oxa/Alb3 family includes a group of conserved translocases that are essential for protein insertion into inner membranes of bacteria and mitochondria, and thylakoid membranes of chloroplasts. Because mitochondria and chloroplasts are of bacterial origin, Oxa and Alb3, like many other mitochondrial/chloroplastic proteins, are hypothetically derived from the pre-existing protein (YidC) of bacterial endosymbionts. Here, we test this hypothesis and investigate the evolutionary history of the whole YidC/Oxa/Alb3 family in the three domains of life.     

Defining the regions of Escherichia coli YidC that contribute to activity

The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.     

Diatom fucoxanthin chlorophyll a/c-binding protein (FCP) and land plant light-harvesting proteins use a similar pathway for thylakoid membrane Insertion

The light-harvesting proteins in plastids of different lineages including algae and land plants represent a superfamily of chlorophyll-binding proteins that seem to be phylogenetically related, although some of the light-harvesting complex (LHC) proteins bind different carotenoids. LHCs can be divided into chlorophyll a/b-binding proteins found in green algae, euglenoids, and higher plants and into chlorophyll a/c-binding proteins of various algal taxa. LHC proteins from diatoms are named fucoxanthin-chlorophyll a/c-binding proteins (FCP). In contrast to chlorophyll a/b-binding proteins, there is no information so far about the way FCPs integrate into thylakoid membranes. The diatom FCP preproteins have a bipartite presequence that is necessary to enable transport into the four membrane-bound diatom plastids, but similar to chlorophyll a/b-binding proteins there is apparently no presequence present for targeting to the thylakoid membrane. By establishing an in vitro import assay for diatom thylakoids, we demonstrated that thylakoid integration of diatom FCP depends on the presence of stromal factors and GTP. This indicates that a pathway involving signal recognition particles (SRP) is involved in membrane integration just as shown for LHCs in higher plants. We also demonstrate integration of diatom FCP into thylakoids of higher plants and vice versa SRP-dependent targeting of LHCs from pea and Arabidopsis into diatom thylakoids. The similar SRP-dependent modes of thylakoid integration of land plant LHCs and FCPs support recent analyses indicating a common origin of chlorophyll a/b- and a/c-binding proteins.     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.     

Sec/SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase

Previously, the role of YidC in the membrane protein biogenesis of the F(0) sector of the Escherichia coli F(1)F(0) ATP synthase was investigated. Whereas subunits a and c of the F(1)F(0) ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F(0) sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH(2)-terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.     

YidC is strictly required for membrane insertion of subunits a and c of the F(1)F(0)ATP synthase and SecE of the SecYEG translocase

YidC was previously discovered to play a critical role for the insertion of the Sec-independent M13 procoat and Pf3 coat phage proteins into the Escherichia coli inner membrane. To determine whether there is an absolute requirement of YidC for membrane protein insertion of any endogenous E. coli proteins, we investigated a few representative membrane proteins. We found that membrane subunits of the F(0) sector of the F(1)F(0)ATP synthase and the SecE protein of the SecYEG translocase are highly dependent on YidC for membrane insertion, based on protease mapping and immunoblot analysis. We found that the SecE dependency on YidC for membrane insertion does not contradict the observation that depletion of YidC does not block SecYEG-dependent protein export at 37 degrees C. YidC depletion does not decrease the SecE level low enough to block export at 37 degrees C. In contrast, we found that protein export of OmpA is severely blocked at 25 degrees C when YidC is depleted, which may be due to the decreased SecE level, as a 50% decrease in the SecE levels drastically affects protein export at the cold temperature [Schatz, P. J., Bieker, K. L., Ottemann, K. M., Silhavy, T. J., and Beckwith, J. (1991) EMBO J. 10, 1749-57]. These studies reported here establish that physiological substrates of YidC include subunits of the ATP synthase and the SecYEG translocase, demonstrating that YidC plays a vital role for insertion of endogenous membrane proteins in bacteria.     

One of two alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas

Proteins of the YidC/Oxa1p/ALB3 family play an important role in inserting proteins into membranes of mitochondria, bacteria, and chloroplasts. In Chlamydomonas reinhardtii, one member of this family, Albino3.1 (Alb3.1), was previously shown to be mainly involved in the assembly of the light-harvesting complex. Here, we show that a second member, Alb3.2, is located in the thylakoid membrane, where it is associated with large molecular weight complexes. Coimmunoprecipitation experiments indicate that Alb3.2 interacts with Alb3.1 and the reaction center polypeptides of photosystem I and II as well as with VIPP1, which is involved in thylakoid formation. Moreover, depletion of Alb3.2 by RNA interference to 25 to 40% of wild-type levels leads to a reduction in photosystems I and II, indicating that the level of Alb3.2 is limiting for the assembly and/or maintenance of these complexes in the thylakoid membrane. Although the levels of several photosynthetic proteins are reduced under these conditions, other proteins are overproduced, such as VIPP1 and the chloroplast chaperone pair Hsp70/Cdj2. These changes are accompanied by a large increase in vacuolar size and, after a prolonged period, by cell death. We conclude that Alb3.2 is required directly or indirectly, through its impact on thylakoid protein biogenesis, for cell survival.     

Signal recognition particle-dependent protein targeting, universal to all kingdoms of life

The signal recognition particle (SRP) and its membrane-bound receptor represent a ubiquitous protein-targeting device utilized by organisms as different as bacteria and humans, archaea and plants. The unifying concept of SRP-dependent protein targeting is that SRP binds to signal sequences of newly synthesized proteins as they emerge from the ribosome. In eukaryotes this interaction arrests or retards translation elongation until SRP targets the ribosome-nascent chain complexes via the SRP receptor to the translocation channel. Such channels are present in the endoplasmic reticulum of eukaryotic cells, the thylakoids of chloroplasts, or the plasma membrane of prokaryotes. The minimal functional unit of SRP consists of a signal sequence-recognizing protein and a small RNA. The as yet most complex version is the mammalian SRP whose RNA, together with six proteinaceous subunits, undergo an intricate assembly process. The preferential substrates of SRP possess especially hydrophobic signal sequences. Interactions between SRP and its receptor, the ribosome, the signal sequence, and the target membrane are regulated by GTP hydrolysis. SRP-dependent protein targeting in bacteria and chloroplasts slightly deviate from the canonical mechanism found in eukaryotes. Pro- and eukaryotic cells harbour regulatory mechanisms to prevent a malfunction of the SRP pathway. Electronic Publication