Browsing by Subject "Proteintransport"

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Functional and structural studies of a C-terminally extended YidC
(2015) Seitl, Ines; Kuhn, Andreas
Members of the YidC/Oxa1/Alb3 protein family catalyze the insertion of integral membrane proteins into the lipid bilayer of the bacterial plasma membrane (YidC), the inner mitochondrial membrane (Oxa1), and the chloroplast thylakoid membrane (Alb3) (Saller et al., 2012; Dalbey et al., 2014). The insertase homologs are comprised of a conserved core region of 5 transmembrane domains, but are provided with additionally flanking N- and C-terminal regions of variable lengths and functions. The Gram-negative YidC is characterized by an additional N-terminal domain, while Gram-positive bacteria, mitochondria and plastids developed C-terminally extended insertase-domains. These domains are involved e.g. in direct interaction with ribosomes and facilitate a functional overlap with the co-translational SRP-targeting pathway. An extended C-terminal highly positively charged tail region was also found in the YidC homologs of the Gram-negative marine bacteria Rhodopirellula baltica and Oceanicaulis alexandrii, but not in Escherichia coli. The primary subject of this work was to characterize and analyze in detail the C-terminally extended YidC chimera, composed of the E. coli YidC and the C-terminally extended domains of the marine YidC homologs. Biochemical binding assays with the purified YidC proteins and isolated, vacant E. coli 70S ribosomes showed that the C-tails mediate specific binding to ribosomes independently of the translational state of the ribosome. Furthermore, a ribosome-bound insertase complex was visualized by cryo-electron microscopy. The enhanced affinity of the C-terminally extended YidC was used to isolate stable complexes with stalled ribosomes, carrying a nascent polypeptide chain of a YidC substrate protein (MscL). The cryo-EM structure of a YidC-ribosome nascent chain complex (RNC) was solved to a 8,6 Å resolution and allowed the visualization of the nascent chain from the peptidyl transferase center through the ribosomal exit tunnel into the YidC density. The structure revealed the helix H59 of the 23S rRNA and the two ribosomal proteins L24 and L29 as the major contacts sites of YidC at the ribosomal tunnel exit. Pull down assays confirmed a significantly interaction of the C-terminal ribosome binding domain and the ribosomal protein L29, while L24 seems to be a universal contact site for the YidC-insertase core domain. Strikingly, the cryo-EM structure clearly showed a single monomer of YidC bound to the translating ribosome. This suggests that monomeric YidC might be the minimal functional unit for YidC-dependent, co-translational insertion of inner membrane proteins. In addition to the in vitro tests, a possible role of the C-terminal YidC extensions in co-translational protein targeting was tested in vivo in E. coli. For that purpose the targeting and localization of the SRP-dependent YidC-substrate protein MscL (Facey et al., 2007) was investigated as a GFP fusion protein via fluorescence microscopy. In addition, the proper membrane insertion of MscL was analyzed in radioactive pulse chase experiments via AMS gel shift assays, either in the absence of a functional SRP or SRP receptor (FtsY). Both in vivo assays clearly showed that the C-terminal ribosome binding domain of the R. baltica YidC homolog can partially substitute for the SRP receptor function in E. coli, while the cytosolic signal recognition particle is still required for correct insertion of the MscL protein. Therefore, a new co-translational targeting and insertion model of YidC-only substrates was proposed. This works also highlights evolutionary aspects of the accessory YidC domains and indicates that the C-terminal extended tail of YidC in the planctomycete group may be an ancestral remnant of a primordial translocation system operating without a typical SRP receptor. The second part focuses on the interaction of the signal recognition particle with SRP signal sequences. Isolated mutant signal sequence peptides were used to determine the specificity of SRP recognition in proteins. The interaction studies were established in an in vitro system and binding affinities of purified SRP to the isolated signal sequence peptides were determined via microscale thermophoresis (MST). A short sequence of 27 amino acid residues at the very N-terminal tail of the large cytoplasmic domain of KdpD was identified as a SRP signal sequence. Furthermore, a direct influence of the amino acid composition in the signal peptide on its SRP binding affinity in vitro was demonstrated. This confirms a low influence of an altered charge in the N-terminal region while mutations in the hydrophobic core region causes significantly reduced binding affinities to SRP. Taken together, this study contributes to the understanding of the molecular mechanisms of co-translational membrane protein biogenesis in bacteria.
Funktion und Dynamik eines gemeinsamen Insertionskomplexes der Sec-Translokase und YidC-Insertase in der bakteriellen Membran
(2020) Steudle, Anja; Kuhn, Andreas
YidC/Oxa1/Alb3-insertases and the Sec-translocase are conserved across all three kingdoms of life and constitute the most important pathway for integral proteins into cell membranes and membranes of eukaryotic organelles. The insertion of membrane proteins into the inner membrane of Gram-negative bacteria occurs mainly via the SecYEG-translocase and the YidC-insertase acting independently or in cooperation. For the cooperative insertion a close contact between SecY and YidC is assumed. Previous interaction-studies and a recently solved low-resolution structure of the so-called holo-translocon (14 Å) indicate a contact between the lateral gate of SecY and the hydrophobic substrate slide of YidC. Which specific domains of YidC and SecY thereby interact directly with each other was unknown so far. The aim of this study was to describe the contact between SecY and YidC in more detail. A high affinity for the interaction of the two proteins in detergent and in DOPC-proteoliposomes was determined via FRET measurements with fluorescently labeled SecY and YidC. For the stoichiometric ratio of the SecY/YidC-interaction a factor of one was calculated. To identify the specific contacts between SecY and YidC in vivo disulphide cross-linking experiments were performed. Direct interactions between the transmembrane domain (TM) 3 and TM8 of the SecY lateral gate and TM3 and TM5 of the hydrophobic slide of YidC were found, respectively. Furthermore, a YidC mutant with five serine substitutions, which was unable to rescue a YidC depletion strain, was investigated. Even though the serine positions are located in the middle and the periplasmic half of the hydrophobic slide of YidC and four of the positions are identical with substrate contact sites, no inhibition of insertion for the YidC-dependent substrates M13 procoat and Pf3 coat by the 5S mutant compared to the wildtype YidC was observed. For the YidC-only pathway a minimum of hydrophobicity seems to be required sufficient to allow the insertion of these substrates. In vitro FRET measurements showed an impaired interaction between SecY and the YidC 5S mutant and confirmed once again an involvement of the hydrophobic slide in the SecY/YidC-contact. Based on the cross-linking contacts and the results of the FRET measurements a possible model of the SecY/YidC-contact was established, which shows the SecY lateral gate vis-à-vis of the hydrophobic slide and the hydrophilic groove between TM3 and TM5 of YidC generating a combined SecY/YidC-cavity. Taken together, the present study provides further evidence that the lateral gate of the Sec-translocase directly interacts with the hydrophobic slide of YidC. In a further project, a SecY-YidC fusion protein was cloned to ensure the two proteins are in close proximity, the correct orientation and proper stoichiometry after reconstitution into proteoliposomes. For a collaboration with the ETH Zürich, proteoliposomes hosting the fusion protein, SecYEG, YidC or SecYEG and YidC together were prepared by myself in Hohenheim. The stepwise insertion of the Sec/YidC-dependent substrate LacY into these proteoliposomes was observed by a collaborating group of the ETH Zürich using AFM–based single-molecule force spectroscopy. The insertion of LacY was observed for the different cases but for the fusion protein and SecYEG combined with YidC the insertion process is dominated by the Sec-translocase, whereas YidC probably only has a supporting function in the folding of the protein.
Die Insertion des „minor coat“ Proteins G3P des Bakteriophagen M13 in die innere E. coli Membran benötigt die Insertase YidC und die Translokase SecYEG.
(2021) Kleinbeck, Farina; Kuhn, Andreas
The membrane of every cell forms a spacial limitation for this smallest unit of a life form. Such a very simple unicellular life form is also the Gram-negative bacterium Escherichia coli (E. coli) and is therefore a valid model organism for a living cell. Due to the inner membrane the cellular components are held together in close proximity and are separated from the extracellular environment. Most substrates cannot pass the lipid bilayer, which forms the membrane, so an import and export system had to be developed to accomplish this. For these import and export systems, very complex, polytopic transmembrane protein complexes are needed. Examples are ion channels, ion pumps or large complexes through which energy production, secretion of toxins and the transfer of nutrients are catalysed. Moreover, proteins with functions in the periplasm or outer membrane must also travel from their site of synthesis in the cytoplasm to their destination. For these different processes proteins must be inserted into or translocated across the inner membrane. Of the total proteome in prokaryotes approximately 25 to 30% is either inserted into or secreted across the inner membrane. This work identified several components required for the insertion of the "minor coat" protein G3P of M13 bacteriophage. This protein is important for the assembly of the phage particle that occurs in the inner membrane. The outermost C-terminus of G3P is anchored in the inner membrane via a single transmembrane domain, while the bulk of the approximately 42 kDa protein is located in the periplasm. Using an N-terminal cleavable signal peptide, the major portion of G3P is translocated into the periplasm via SecYEG with the help of SecA and the membrane potential. Targeting, on the other hand, could not be clearly assigned to one of the known post- or co-translational pathways. Although contact via disulfide crosslink studies to Ffh, the protein component of the ribonucleoprotein SRP, was observed via stalled ribosome nascent chains (RNCs), insertion into the membrane in vivo was independent of Ffh. Even when the interaction between SecY and FtsY, the receptor for SRP at the membrane, was impaired, G3P was inserted via SecYEG. Although the chaperone SecB was able to bind to G3P in vitro, G3P inserted independently of SecB in vivo. For membrane incorporation of G3P, it was shown that YidC is required in vivo in addition to SecYEG. Disulphide crosslink studies demonstrated that G3P first contacts the plug domain TM2b and lateral gate (TM2a and TM7) via the signal peptide of G3P, and finally the C-terminal transmembrane domain of G3P contacts YidC via TM3 and TM5 of the hydrophilic slide. Based on these contact sites, a possible insertion model was confirmed, with SecY and YidC mediating defined steps in the insertion process, providing new insights into this largely unknown process.
Membrane targeting and insertion of the sensor protein KdpD and the C-tail anchored protein SciP of Escherichia coli
(2019) Proß, Eva; Kuhn, Andreas
In E. coli, most inner membrane proteins are targeted in a co-translational manner by the universally conserved signal recognition particle (Bernstein et al. 1989; Valent et al. 1998; Schibich et al. 2016). SRP scans the translating ribosomes and binds with high affinity to an exposed SRP signal sequence, present in the nascent chain (Bornemann et al. 2008; Holtkamp et al. 2012; Saraogi et al. 2014). After targeting to the membrane-associated SRP receptor FtsY, the nascent membrane protein is forwarded to the Sec translocase or to the YidC insertase to be integrated into the bilayer (Miller et al. 1994; Cross et al. 2009; Welte et al. 2012; Akopian et al. 2013). In general, the targeting and insertion pathways of inner membrane proteins in E. coli are already well studied. However, there is a special class of proteins, the C-tail anchored proteins with only a few members in E. coli, whose insertion mechanisms are unknown in prokaryotes to date. To study those insertion mechanisms, the C-tail anchored protein SciP was used as a model protein. SciP from the enteroaggregative E. coli is a structural component of the type 6 secretion system and contains a transmembrane domain (TMD) at the extreme C-terminal part from amino acid 184 to 206. This results in a large N-terminal cytoplasmic domain of 183 amino acids. In E. coli, there is another protein, the potassium sensor protein KdpD which shares with SciP the commonality of a large N-terminal cytoplasmic domain. KdpD is a four-spanning membrane protein with the first TMD starting at amino acid position 400. For both proteins, with the TMD being located far away from the cytoplasmic N-terminal part, it was thought that they cannot use the co-translational SRP pathway. However, it was shown that KdpD is targeted co-translationally by SRP and a cytoplasmic targeting signal located between amino acids 22-48 was identified (Maier et al. 2008). In this study it was shown that the C-tail anchored protein SciP is also targeted early during translation by SRP. With fluorescence microscopy studies and sfGFP-SciP fusion constructs, two short hydrophobic regions in the N-terminal cytoplasmic domain (amino acids 12-20 and 62-71) were identified as being important for membrane targeting. With artificially stalled ribosomes exposing each of the targeting signal, microscale thermophoresis meausurements decoded that both signals bind to SRP and to a preincubated SRP-FtsY complex, mimicking the next targeting step. Cysteine-accessibilty assays demonstrated that SciP is the first described protein with two targeting signals since the deletion of one of the hydrophobic regions was compensated by the other remaining one in vivo. To decipher the crucial features of the novel cytoplasmic SRP signal sequences of KdpD and SciP alterations in the signal sequences were analyzed with fluorescence microscopy using sfGFP fusion constructs and microscale thermophoresis measurements using stalled ribosomes. These studies revealed that the novel signal sequences have to exceed a threshold level of hydrophobicity to be recognized and bound by SRP and target sfGFP to the membrane. In addition, three positively charged amino acids in the KdpD SRP signal sequence were identified to promote SRP binding. To characterize the binding mechanism of SRP to the signal sequences, in vitro disulphide cross-linking studies with synthesized KdpD22-48, SciP1-27 and SciP54-85 peptides were performed. All three peptides could be cross-linked to the hydrophobic groove of SRP formed by the M domain, which correlates with the binding of SRP to other substrates. Taken together, the results show that SRP binding is not limited to the TMDs of proteins. SRP is also able to recognize short hydrophobic stretches in the cytoplasmic domain of inner membrane proteins. Cysteine-accessibility assays with the C-tail anchored protein SciP decoded that not only SRP is involved in the delivery pathway but also the insertase YidC. With only 11 amino acids in the periplasmic domain SciP matches with the characteristics of other known YidC only substrates. By extending the C-tail of SciP it was found out that a critical length of 20 amino acids exists and that the exceed of this limit makes the insertion of SciP dependent on the Sec translocase. The studies with the extended C-tails of SciP helped to gain more general information about the YidC dependent insertion of proteins. The results obtained with the protein SciP are first indications about how the insertion of C-tail anchored proteins occurs in E. coli. It is assumed that the SRP system and the insertase YidC compensate the absence of the eukaryotic Get system, responsible for the insertion of eukaryotic tail-anchored proteins.
The bacterial membrane insertase YidC : in vivo studies of substrate binding and membrane insertion
(2015) Klenner, Christian Daniel; Kuhn, Andreas
YidC of Escherichia coli belongs to the evolutionarily conserved proteins of the Oxa1/YidC/Alb3 insertase family. The transmembrane regions of the core domain, comprising of TM2-6, are the most conserved parts among the homologs and are crucial for the function as a membrane insertase. This is particularly true for the TM2, TM3 and TM5 (KUHN et al., 2003; KIEFER & KUHN, 2007). In bacteria, YidC acts as an independently working membrane insertase and, as well, in cooperation with the Sec translocon for the biogenesis of various membrane proteins. YidC is required for the biogenesis of respiratory complexes, ATP synthase and for example the mechanosensitive channel protein MscL. Also, the coat proteins of filamentous phage Pf3 and M13 require YidC for membrane insertion. The best studied substrate is the Pf3 coat protein of phage Pf3 infecting Pseudomonas aeruginosa – i.e. a small protein of 44 amino acids in length. In the context of this thesis, the YidC-dependent biogenesis of Pf3 coat was analyzed to gain better insight into the entire insertion process. In doing so, a set of more than 100 single cysteine mutants in distinct domains of YidC and Pf3 coat were generated. To study the insertion of Pf3 coat under physiological conditions, an in vivo cross-linking assay was established for capturing YidC-Pf3 interactions within a short period of time after the onset of synthesis (1 minute) using 35S-Met pulse-labelling methods. YidC binds inserting Pf3 coat protein in distinct regions of the highly conserved TM domains involving four of the six TM helices. It was verified that TM3 is indispensable for the function of YidC since four contacting residues were found in this TM helix. A helical wheel projection of substrate binding helices reveals the localization of the contacting residues of each TM segment on one helical face. This implies a helix arrangement of the transmembrane core domain which enables binding of inserting substrate proteins and interactions with transmembrane domains over the entire membrane-spanning part of YidC. The serial mutation of nine from twelve contacting residues, which are strongly hydrophobic in most cases, to serines impaired the function of YidC, whereas the single mutations had no effect. Additionally, the insertion process of translocation deficient Pf3 coat mutants was analyzed for intermediate states of the insertion process. It has been shown that the insertion deficient Pf3 coat mutants are inhibited at a late step of membrane insertion, i.e. forming the YidC contacts in the periplasmic leaflet. Based on this work, further studies confirmed that the identified substrate contacting regions of YidC play a key role in YidC-mediated insertion. The mechanosensitive channel protein MscL, M13 procoat, nascent Foc and the polytopic membrane protein LacY contact YidC at exactly the same positions (NEUGEBAUER et al., 2012; SPANN & KUHN, unpublished results; WICKLES et al., 2014; ZHU et al., 2013b).