Import Of Preproteins Research Articles

Nucleotides and Substrates Trigger the Dynamics of the Toc34 GTPase Homodimer Involved in Chloroplast Preprotein Translocation

GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases.

From inventory to functional mechanisms

For decades, the pyruvate dehydrogenase complex in the mitochondrial matrix was considered as a rare example of how protein kinases and phosphatases can regulate important functions within this organelle. During the last decade, several proteomic studies revealed that a large fraction of mitochondrial proteins are indeed phosphorylated. A surprisingly high number of phosphorylation sites was found at the preprotein import machinery, TOM, in the outer membrane that provides the central protein import gate for most mitochondrial precursors synthesized in the cytosol. This review describes current knowledge of the mitochondrial phosphoproteome and introduces the first regulatory mechanisms of protein import dynamics by reversible phosphorylation, which have been uncovered mainly in the model organism Saccharomycescerevisiae.

In Vivo Function of Tic22, a Protein Import Component of the Intermembrane Space of Chloroplasts

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.

Three old and one new: protein import into red algal-derived plastids surrounded by four membranes

Engulfment of a red or green alga by another eukaryote and subsequent reduction of the symbiont to an organelle, termed a complex plastid, is a process known as secondary endosymbiosis and is shown in a diverse group of eukaryotic organisms. Important members are heterokontophytes, haptophytes, cryptophytes, and apicomplexan parasites, all of them with complex plastids of red algal origin surrounded by four membranes. Although the evolutionary relationship between these organisms is still debated, they share common mechanisms for plastid protein import. In this review, we describe recent findings and current models on preprotein import into complex plastids with a special focus on the second outermost plastid membrane. Derived from the plasma membrane of the former endosymbiont, the evolution of protein transport across this so-called periplastidal membrane most likely represented the challenge in the transition from an endosymbiont to a host-dependent organelle. Here, remodeling and relocation of the symbiont endoplasmic reticulum-associated degradation (ERAD) machinery gave rise to a translocon complex termed symbiont-specific ERAD-like machinery and provides a fascinating insight into complex cellular evolution.

Biophysical Characterization and Conformational Analysis of the GTPase Domain of Pre-Protein Import Receptors atToc33G and psToc34G

atToc33 and psToc34 are GTPases-, that act as receptors for pre-protein import in the chloroplast outer envelope. Various lines of evidence suggest that the GTPase domain of atToc33 (atToc33G) dimerizes, including native PAGE studies (Weibel et al., 2003), gel-filtration experiments (Yeh et al., 2007) and filter binding assays (Koenig et al., 2008). The crystal structure of atToc33 shows that it is a monomer (Koenig et al., 2008) while psToc34 crystallizes as a dimer (Sun et al., 2002). The R130A atToc33 mutant apparently leads to monomer formation in vitro (Reddick et al., 2007; Weibel et al., 2003). Both-, atToc33-wt and atToc33-R130A bind GTP and GDP with high affinity and hydrolyse GTP with similar efficiencies (Weibel et al., 2003). To further investigate the conformations of these GTPases, and to characterize their optical spectroscopic properties, we first, expressed atToc33G and its R130A mutant in high yield. Initial characterization of these proteins by CD spectroscopy revealed significant α-helical content in their secondary structure. We also examined the effect of an increase in protein concentration of atToc33G and its R130A mutant using CD and fluorescence spectroscopy. CD spectral line shape does not change with increasing protein concentrations indicating that no or only small conformational changes occurs as the concentration increases. Fluorescence spectra for both proteins are superimposable with an intensity maximum at ∼320nm. The addition of GDP to both the proteins leads to reduction of intensity. No wavelength shift in emission spectrum was observed. Our spectroscopic results and anisotropy experiments show that both atToc33G and atToc33G R130A are monomeric, irrespective of the protein concentration and also in presence of GDP or GTP. Experiments to compare atToc33G and atToc33G R130A with their corresponding homologues from Pisumsativum are also presented.

Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria

Loss of expression of the methylation-controlled J gene, MCJ (DNAJC15), is observed in cases of several tumors and plays a crucial role in the chemoresistance of ovarian cancer cells. Aside from the pathophysiological effects, almost nothing is known about the cellular function of MCJ. Here, we provide the first evidence that MCJ acts in the biogenesis of mitochondria. Our results demonstrate that MCJ is located in mitochondria. It is anchored in the mitochondrial inner membrane with the C-terminal J domain facing the matrix space. We show that MCJ forms a stable subcomplex with a component of the mitochondrial import motor, MAGMAS, a protein overexpressed in cells treated with granulocyte-macrophage colony-stimulating factor and in prostate carcinomas. In addition, MCJ and MAGMAS interact with the core components of the TIM23 pre-protein translocase. We demonstrate that the recombinant soluble MCJ domain stimulates the ATPase activity of the human mtHsp70 chaperone, mortalin, the central component of the import motor of the TIM23 translocase. This stimulation is counteracted by MAGMAS. Moreover, pre-protein import into mitochondria is impaired in the absence of MCJ. Interestingly, MCJ is able to take over the function of Tim14, the essential J co-chaperone of the mitochondrial protein import motor in yeast. In summary, our results show that MCJ functions as J co-chaperone of the human TIM23 pre-protein translocase, suggesting a link between mitochondrial pre-protein import and tumorigenesis.

Phagocytosis of algal chloroplasts by digestive gland cells in the photosynthesis-capable slug Elysia timida (Mollusca, Opisthobranchia, Sacoglossa)

Parapodia of the sacoglossan slug Elysia timida were preserved by high-pressure cryofixation during feeding experiments and investigated with transmission electron microscopy. This slug has been known for its long-term retention of active chloroplasts and photosynthesis. We observed different stages of phagocytosis of chloroplast components from ingested algal food by slug digestive gland cells. Thylakoid stacks and stroma of chloroplasts were engulfed by the slug cells. In the slug cells thylakoids were surrounded by one membrane only. This membrane is interpreted as having been generated by the mollusk during phagocytosis. It is inferred to be eukaryotic in origin and unlikely, therefore, to be endowed with the translocons system ordinarily regulating import of algal gene-encoded plastid preproteins. Our structural findings suggest that chloroplast components in the slug cells are thylakoid stacks with chloroplast stroma only.

Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation

Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The symbiont-derived ERAD-like machinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum. The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc1 complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23(CORE), revealing a binding chain of TIM23(CORE)-Mgr2/Tim21-respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.

Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism

The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.

Chloroplast Omp85 proteins change orientation during evolution

The majority of outer membrane proteins (OMPs) from gram-negative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using self-assembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

Mitochondria breathe for autophagy

Proper mitochondrial structure and function are established by continuous cycles of fusion and fission, and a unidirectional pathway of autophagy‐mediated degradation. In this issue of The EMBO Journal , Graef and Nunnari (2011) demonstrate that autophagy requires healthy mitochondria in cells under amino‐acid starvation. Defects in mitochondrial respiration cause activation of cAMP‐dependent protein kinase A (PKA), an evolutionarily conserved regulator that inhibits autophagic flux. This study reveals a crucial interplay between mitochondrial function and autophagy, and raises the possibility that deregulation in one of these processes simultaneously compromises the other to potentiate risks for various human disorders.

Dimerization of TOC receptor GTPases and its implementation for the control of protein import into chloroplasts

Pre-protein import into chloroplasts is facilitated by multiprotein translocon complexes in the envelope membranes. Major components of the TOC (translocon at the outer envelope membrane of chloroplasts) complex are the receptor proteins Toc33 and Toc159. These two receptors are related GTPases, and they are predicted to engage in homodimerization and/or heterodimerization. Although such dimerization has been studied extensively, its exact function in vivo remains elusive. In this issue of the Biochemical Journal, Oreb et al. present evidence that homodimerization of Toc33 prevents nucleotide exchange, thereby locking the receptor in the GDP-loaded state and preventing further activity. Pre-protein arrival is proposed to release this lock, through disruption of the dimer and subsequent nucleotide exchange. The Toc33-bound pre-protein is then able to progress to downstream steps in the translocation mechanism, with GTP hydrolysis defining another important control point as well as preparing the receptor for the next pre-protein client. These new results are discussed in the context of previous findings pertaining to TOC receptor dimerization and function.

High TIMM17A expression is associated with adverse pathological and clinical outcomes in human breast cancer

Mitochondrial dysfunction can be associated with genomic instability and has been implicated in the pathogenesis of several human malignancies, including breast cancer (BC). The mitochondrial protein, translocase of inner mitochondrial membrane 17 homolog A (TIMM17A) contributes to a pre-protein import complex, essential for mitochondrial function. In this study, TIMM17A mRNA expression was evaluated in benign and malignant breast tissues and correlated with pathological and clinical outcomes. Breast cancer tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription, transcript levels were determined by using real-time quantitative PCR and normalized against CK19. Transcript levels were compared and then analysed against tumour size, tumour grade, oestrogen receptor (ER) status, nodal involvement, TNM stage, Nottingham prognostic index (NPI) and clinical outcome over a 10-year follow-up period. Compared to normal tissue (mean transcript level = 12.7), TIMM17A mRNA expression was higher in BC (62, p = 0.006), TNM-1 (37.9, p = 0.05), TNM-2 (114, p = 0.034), NPI-2 (81, p = 0.041), patients with progressive disease (169, p = 0.017) and those who died from BC (179, p = 0.026). Expression increased with tumour grade; grade 1 versus 2 (12.1 vs.70, p = 0.007), grade 1 versus 3 (12.1 vs.73, p = 0.065, [not significant] NS) and grade 1 versus 2 and 3 (12.1 vs. 72, p = 0.0048). Higher transcript levels were associated with ER-α positivity (94 vs. 31.6, p = 0.073, NS) and ER-β negativity (82 vs. 16, p = 0.015). Nodal positivity was significantly associated with higher transcript levels (101 vs. 28.1, p = 0.046). Compared to disease-free patients (mean transcript level = 24.6), TIMM17A expression was significantly higher in those with progressive disease and patients who died of BC (179, p = 0.037). Higher transcript levels were significantly associated with poorer overall survival after a median follow-up of 10 years (p = 0.010). TIMM17A expression emerged as a strong independent predictor of overall survival in multivariate analysis (p = 0.033). TIMM17A mRNA expression is significantly associated with unfavourable pathological parameters including tumour grade, nodal positivity, TNM stage and NPI; in addition to adverse clinical outcomes such as progressive disease, disease-free and overall survival. TIMM17A offers utility as a prognostic marker and a novel mitochondrial target for potential therapeutic strategies.

The physical and functional borders of transit peptide-like sequences in secondary endosymbionts

BackgroundPlastids rely on protein supply by their host cells. In plastids surrounded by two membranes (primary plastids) targeting of these proteins is facilitated by an N-terminal targeting signal, the transit peptide. In secondary plastids (surrounded by three or four membranes), transit peptide-like regions are an essential part of a bipartite topogenic signal sequence (BTS), and generally found adjacent to a N-terminally located signal peptide of the plastid pre-proteins. As in primary plastids, for which no wealth of functional information about transit peptide features exists, the transit peptide-like regions used for import into secondary ones show some common features only, which are also poorly characterized.ResultsWe modified the BTS (in the transit peptide-like region) of the plastid precursor fucoxanthin-chlorophyll a/c binding protein D (FcpD) fused to GFP as model substrate for the characterization of pre-protein import into the secondary plastids of diatoms. Thereby we show that (i) pre-protein import is highly charge dependent. Positive net charge is necessary for transport across the plastid envelope, but not across the periplastid membrane. Acidic net charge perturbs pre-protein import within the ER. Moreover, we show that (ii) the mature domain of the pre-protein can provide intrinsic transit peptide functions.ConclusionsOur results indicate important characteristics of targeting signals of proteins imported into secondary plastids surrounded by four membranes. In addition, we show a self-targeting mechanism, in which the mature protein domain contributes to the transit peptide function. Thus, this phenomenon lowers the demand for pre-sequences evolved during the course of endosymbiosis.

Effect of denervation-induced muscle disuse on mitochondrial protein import

This study determined whether muscle disuse affects mitochondrial protein import and whether changes in protein import are related to mitochondrial content and function. Protein import was measured using a model of unilateral peroneal nerve denervation in rats for 3 (n = 10), 7 (n = 12), or 14 (n = 14) days. We compared the import of preproteins into the matrix of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria isolated from the denervated and the contralateral control tibialis anterior muscles. Denervation led to 50% and 29% reductions in protein import after 14 days of disuse in SS and IMF mitochondria, respectively. This was accompanied by significant decreases in mitochondrial state 3 respiration, muscle mass, and whole muscle cytochrome c oxidase activity. To investigate the mechanisms involved, we assessed disuse-related changes in 1) protein import machinery components and 2) mitochondrial function, reflected by respiration and reactive oxygen species (ROS) production. Denervation significantly reduced the expression of translocases localized in the inner membrane (Tim23), outer membrane (Tom20), and mitochondrial heat shock protein 70 (mtHsp70), especially in the SS subfraction. Denervation also resulted in elevated ROS generation, and exogenous ROS was found to markedly reduce protein import. Thus our data indicate that protein import kinetics are closely related to alterations in mitochondrial respiratory capacity (r = 0.95) and are negatively impacted by ROS. Deleterious changes in the protein import system likely facilitate the reduction in mitochondrial content and the increase in organelle dysfunction (i.e., increased ROS production and decreased respiration) during chronic disuse, which likely contribute to the activation of degradative pathways leading to muscle atrophy.

A Functional Tom70 in the Human Parasite Blastocystis sp.: Implications for the Evolution of the Mitochondrial Import Apparatus

Core proteins of mitochondrial protein import are found in all mitochondria, suggesting a common origin of this import machinery. Despite the presence of a universal core import mechanism, there are specific proteins found only in a few groups of organisms. One of these proteins is the translocase of outer membrane 70 (Tom70), a protein that is essential for the import of preproteins with internal targeting sequences into the mitochondrion. Until now, Tom70 has only been found in animals and Fungi. We have identified a tom70 gene in the human parasitic anaerobic stramenopile Blastocystis sp. that is neither an animal nor a fungus. Using a combination of bioinformatics, genetic complementation, and immunofluorescence microscopy analyses, we demonstrate that this protein functions as a typical Tom70 in Blastocystis mitochondrion-related organelles. Additionally, we identified putative tom70 genes in the genomes of other stramenopiles and a haptophyte, that, in phylogenies, form a monophyletic group distinct from the animal and the fungal homologues. The presence of Tom70 in these lineages significantly expands the evolutionary spectrum of eukaryotes that contain this protein and suggests that it may have been part of the core mitochondrial protein import apparatus of the last common ancestral eukaryote.

Identification and Characterization of Mitochondrial Targeting Sequence of Human Apurinic/Apyrimidinic Endonuclease 1

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289-318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.

Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency.

The atToc33 protein is one of several pre-protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis-related pre-proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild-type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159-deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.