Browsing by Subject "Microscale Thermophorese"

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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.
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.