Protein Insertion Machinery Research Articles

The intracellular distribution of the components of the GET system in vascular plants

The guided entry of tail-anchored proteins (GET) pathway facilitates targeting and insertion of tail-anchored proteins into membranes. In plants, such a protein insertion machinery for the endoplasmic reticulum as well as constituents within mitochondrial and chloroplasts were discovered. Previous phylogenetic analysis revealed that Get3 sequences of Embryophyta form two clades representing cytosolic (“a”) and organellar (“bc”) GET3 homologs, respectively. Cellular fractionation of Arabidopsis thaliana seedlings and usage of the self-assembly GFP system in protoplasts verified the cytosolic (ATGet3a), plastidic (ATGet3b) and mitochondrial (ATGet3c) localization of the different homologs. The identified plant homologs of Get1 and Get4 in A. thaliana are localized in ER and cytosol, respectively, implicating a degree of conservation of the GET pathway in A. thaliana. Transient expression of Get3 homologs of Solanum lycopersicum, Medicago × varia or Physcomitrella patens with the self-assembly GFP technique in homologous and heterologous systems verified that multiple Get3 homologs with differing subcellular localizations are common in plants. Chloroplast localized Get3 homologs were detected in all tested plant systems. In contrast, mitochondrial localized Get3 homologs were not identified in S. lycopersicum, or P. patens, while we confirmed on the example of A. thaliana proteins that mitochondrial localized Get3 proteins are properly targeted in S. lycopersicum as well.

Plant Mitochondrial Inner Membrane Protein Insertion.

During the biogenesis of the mitochondrial inner membrane, most nuclear-encoded inner membrane proteins are laterally released into the membrane by the TIM23 and the TIM22 machinery during their import into mitochondria. A subset of nuclear-encoded mitochondrial inner membrane proteins and all the mitochondrial-encoded inner membrane proteins use the Oxa machinery—which is evolutionarily conserved from the endosymbiotic bacterial ancestor of mitochondria—for membrane insertion. Compared to the mitochondria from other eukaryotes, plant mitochondria have several unique features, such as a larger genome and a branched electron transport pathway, and are also involved in additional cellular functions such as photorespiration and stress perception. This review focuses on the unique aspects of plant mitochondrial inner membrane protein insertion machinery, which differs from that in yeast and humans, and includes a case study on the biogenesis of Cox2 in yeast, humans, two plant species, and an algal species to highlight lineage-specific similarities and differences. Interestingly, unlike mitochondria of other eukaryotes but similar to bacteria and chloroplasts, plant mitochondria appear to use the Tat machinery for membrane insertion of the Rieske Fe/S protein.

Abstract 4412: c-Myc-driven lymphomas require calcium-modulating cyclophilin ligand (CAML) for survival and proliferation

Abstract Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions in a complex to mediate tail-anchored (TA) protein insertion into the ER membrane. CAML has also been implicated as a regulator of endocytic trafficking, modulator of intracellular calcium signaling, and essential factor for the survival and proliferation of specialized immune cells. To investigate the role of CAML in cell biology and cancer, we generated Eμ-Myc transgenic mice that carry a conditional deletion allele of CAML. From these mice, we derived B cell lymphoma cell lines driven by c-Myc and induced deletion of the CAML gene by treatment with tamoxifen to activate Cre-ERT2 in vitro. Homozygous loss of CAML in multiple independent B cell lymphoma lines activated apoptosis, while heterozygous deletion led to reduced cell proliferation. Apoptosis was inhibited by caspase inhibitor Q-VD-OPh or Bcl-2/xL overexpression; however, blocking apoptosis did not restore cell division. Loss of CAML did not activate the unfolded protein response, but did induce c-Jun and JNK phosphorylation. Structure-function studies indicated that lymphoma cell viability did not depend on interaction of CAML with TRC40, another component of TA protein insertion machinery. Conversely, expression of a minimal region of CAML that interacts with the TA insertion co-factor WRB was sufficient to rescue growth of CAML-deleted cells. In summary, our study demonstrates an essential pro-survival role for CAML in c-Myc-driven lymphomas. Future investigations will focus on the underlying mechanism of apoptosis in this system to advance our understanding of the biological functions of CAML, and consider CAML-dependent survival as a potential novel target for anticancer therapy. Citation Format: Jennifer C. Shing, Lonn D. Lindquist, Richard J. Bram. c-Myc-driven lymphomas require calcium-modulating cyclophilin ligand (CAML) for survival and proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4412.

Investigating Env9's Mechanism of Membrane Insertion in Saccharomyces cerevisiae

Tail‐anchored proteins have vital roles within the cell such as vesicular transport and apoptosis, and often times their specific membrane localization is crucial to their function. These proteins have hydrophobic regions near their C‐terminus, making them identifiable to protein insertion machinery. One pathway which targets C‐tail anchored proteins is the well‐characterized Guided Entry of Tail‐Anchored Protein (GET) complex, consisting of Get1‐5 and Sgt2. Interestingly, a second mechanism has been proposed named the SRP‐independent (SND) pathway which similarly targets tail‐anchored proteins. Our protein of interest, Env9, is one of four novel endosome to vacuole interface (ENV) gene products and our data suggests it possesses a hydrophobic region near its C‐terminus. Env9 localizes to lipid droplet membranes however, its insertion mechanism is unknown. We hypothesize Env9 is targeted to membranes by either GET or SND pathways because of both its c‐terminal hydrophobic domain and its interactions with components of both complexes. Negative genetic interactions with both ENV8 (GET4) and with ENV10 (SND2) have been reported in systematic studies and have been observed in our laboratory. In order to investigate these interactions, we used both microscopic and biochemical approaches. Env9 was tagged with either GFP (green fluorescent protein) under an inducible galactose promoter or HA (hemagglutinin) under a constitutive PGK promoter to analyze localization in GET and SND mutants via confocal microscopy and western blot analysis, respectively. Preliminary microscopy results show slight disruption in GET mutants whereas SND mutants appear to have wild‐type Env9 localization patterns. In preliminary western blot analysis, tagged double mutants Δget4/env9 and Δsnd2/env9 continue to localize Env9‐HA to the P13 pellet that includes lipid droplets, but also vacuoles and aggregated protein complexes. These results suggest Env9 may be targeted to membranes via the GET complex and specifically implicate the GET complex in the process of insertion of Env9 into the ER membrane. These moderate phenotypes may also suggest compensation by one complex in the absence of the other in order to accurately target vital C‐tail anchored proteins. Additionally, the compensatory effect may be further stimulated by the highly overexpressed constructs used in these studies. We are currently exploring the insertion of Env9 expressed at native levels. Additionally, we are examining Env9 localization in GET double and triple mutants, as well as determining protein‐protein interactions via other biochemical approaches such as co‐immunoprecipitation.Support or Funding InformationAcknowledgements: This project was supported by NIH AREA #R‐15 GM85794‐02, NSF‐MRI grant #DBI0722757 for confocal microscopy, and NIH 2R25GM071638‐09A1 for the Research Initiative for Scientific Advancement Fellowship funding. We'd like to thank Dr. Maya Schuldiner at the Weizmann Institute of Science for her donations of the GET and SND mutants.

Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

Partial suppression of Oxa1 mutants by mitochondria‐targeted signal recognition particle provides insights into the evolution of the cotranslational insertion systems

The biogenesis of hydrophobic membrane proteins involves their cotranslational membrane integration in order to prevent their unproductive aggregation. In the cytosol of bacteria and eukaryotes, membrane targeting of ribosomes that synthesize membrane proteins is achieved by signal recognition particles (SRPs) and their cognate membrane-bound receptors. As is evident from the genomes of fully sequenced eukaryotes, mitochondria generally lack an SRP system. Instead, mitochondrial ribosomes are physically associated with the protein insertion machinery in the inner membrane. Accordingly, deletion of ribosome-binding sites on the Oxa1 insertase and the Mba1 ribosome receptor in yeast leads to severe defects in cotranslational protein insertion and results in respiration-deficient mutants. In this study, we expressed mitochondria-targeted versions of the bacterial SRP protein Ffh and its receptor FtsY in these yeast mutants. Interestingly, Ffh was found to bind to the large subunit of mitochondrial ribosomes, and could relieve, to some degree, the defect of these insertion mutants. Although FtsY could also bind to mitochondrial membranes, it did not improve membrane protein biogenesis in this strain, presumably because of its inability to interact with Ffh. Hence, mitochondrial ribosomes are still able to interact physically and functionally with the bacterial SRP system. Our observations are consistent with a model according to which the protein insertion system in mitochondria evolved in three steps. The loss of genes for hydrophilic polypeptides (step 1) allowed the development of ribosome-binding sites on membrane proteins (step 2), which finally made the existence of an SRP-mediated system dispensable (step 3).

Epstein-Barr Viral BNLF2a Protein Hijacks the Tail-anchored Protein Insertion Machinery to Block Antigen Processing by the Transport Complex TAP

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.

The Mechanism of Tail-Anchored Protein Insertion into the ER Membrane

Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 dissociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.

Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR

Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity.

Control of Membrane Protein Topology by a Single C-Terminal Residue

The mechanism by which multispanning helix-bundle membrane proteins are inserted into their target membrane remains unclear. In both prokaryotic and eukaryotic cells, membrane proteins are inserted cotranslationally into the lipid bilayer. Positively charged residues flanking the transmembrane helices are important topological determinants, but it is not known whether they act strictly locally, affecting only the nearest transmembrane helices, or can act globally, affecting the topology of the entire protein. Here we found that the topology of an Escherichia coli inner membrane protein with four or five transmembrane helices could be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C terminus. This observation points to an unanticipated plasticity in membrane protein insertion mechanisms.

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA) [Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA [Ser]Sec to form O-phosphoseryl-tRNA [Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA [Ser]Sec. Sec could be synthesized on tRNA [Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins.

Did the last common ancestor have a biological membrane?

All theories about the origin and evolution of membrane bound cells necessarily have to cope with the nature of the last common ancestor of cellular life. One of the most important aspect of this ancestor, whether it had a closed biological membrane or not, has recently been intensely debated. Having a consensus about it would be an important step towards an eventual (though probably still remote) synthesis of the best elements of the current multitude of cell evolution models. Here I analyse the structural and functional conservation of the few universally distributed proteins that were undoubtedly present in the last common ancestor and that carry out membrane-associated functions. These include the SecY subunit of the protein-conducting channel, the signal recognition particle, the signal recognition particle receptor, the signal peptidase, and the proton ATPase. The conserved structural and functional aspects of these proteins indicate that the last common ancestor was associated with a hydrophobic layer with two hydrophilic sides (an inside and an outside) that had a full-fledged and asymmetric protein insertion and translocation machinery and served as a permeability barrier for protons and other small molecules. It is difficult to escape the conclusion that the last common ancestor had a closed biological membrane from which all cellular membranes evolved.

Evolution of mitochondrial Oxa proteins from bacterial YidC: Inherited and acquired functions of a conserved protein insertion machinery

Evolution of mitochondrial Oxa proteins from bacterial YidC: Inherited and acquired functions of a conserved protein insertion machinery

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria.

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec.

Eukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes.

Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA.

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N-terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N-terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N-termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co-translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.