scholarly journals SecA mediates cotranslational targeting and translocation of an inner membrane protein

2017 ◽  
Vol 216 (11) ◽  
pp. 3639-3653 ◽  
Author(s):  
Shuai Wang ◽  
Chien-I Yang ◽  
Shu-ou Shan
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Inner Membrane ◽  
Nascent Polypeptide ◽  
Inner Membrane Protein

Protein targeting to the bacterial plasma membrane was generally thought to occur via two major pathways: cotranslational targeting by signal recognition particle (SRP) and posttranslational targeting by SecA and SecB. Recently, SecA was found to also bind ribosomes near the nascent polypeptide exit tunnel, but the function of this SecA–ribosome contact remains unclear. In this study, we show that SecA cotranslationally recognizes the nascent chain of an inner membrane protein, RodZ, with high affinity and specificity. In vitro reconstitution and in vivo targeting assays show that SecA is necessary and sufficient to direct the targeting and translocation of RodZ to the bacterial plasma membrane in an obligatorily cotranslational mechanism. Sequence elements upstream and downstream of the RodZ transmembrane domain dictate nascent polypeptide selection by SecA instead of the SRP machinery. These findings identify a new route for the targeting of inner membrane proteins in bacteria and highlight the diversity of targeting pathways that enables an organism to accommodate diverse nascent proteins.

scholarly journals Early encounters of a nascent membrane protein

2005 ◽  
Vol 170 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Edith N.G. Houben ◽  
Raz Zarivach ◽  
Bauke Oudega ◽  
Joen Luirink
Keyword(s):  
Membrane Protein ◽  
Ribosomal Proteins ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Inner Membrane Protein

An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

scholarly journals Structural insights into a novel family of integral membrane siderophore reductases

2021 ◽  
Vol 118 (34) ◽  
pp. e2101952118
Author(s):  
Inokentijs Josts ◽  
Katharina Veith ◽  
Vincent Normant ◽  
Isabelle J. Schalk ◽  
Henning Tidow
Keyword(s):  
Iron Uptake ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Inner Membrane Protein ◽  
Uptake Studies ◽  
Structural Insights

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

scholarly journals In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host

2002 ◽  
Vol 184 (12) ◽  
pp. 3260-3267 ◽  
Author(s):  
R. Wesley Rose ◽  
Mechthild Pohlschröder
Keyword(s):  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sequence Comparisons ◽  
Protein Insertion ◽  
Native Host

ABSTRACT The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

scholarly journals Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

2017 ◽  
Vol 199 (9) ◽  
Author(s):  
Yunxue Guo ◽  
Xiaoxiao Liu ◽  
Baiyuan Li ◽  
Jianyun Yao ◽  
Thomas K. Wood ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Transmembrane Domain ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

scholarly journals A Functional GTPase Domain, but not its Transmembrane Domain, is Required for Function of the SRP Receptor β-subunit

1998 ◽  
Vol 142 (2) ◽  
pp. 341-354 ◽  
Author(s):  
Stephen C. Ogg ◽  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Membrane Protein ◽  
Binding Site ◽  
Protein Targeting ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Single Amino Acid ◽  
Secretory Proteins ◽  
Gtp Binding ◽  
Er Membrane

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRα and SRβ, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRβ. This notion is supported (a) by Srp102p's sequence similarity to SRβ; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRα homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.

scholarly journals The Polybasic Juxtamembrane Region of Sso1p Is Required for SNARE Function In Vivo

2005 ◽  
Vol 4 (12) ◽  
pp. 2017-2028 ◽  
Author(s):  
Jeffrey S. Van Komen ◽  
Xiaoyang Bai ◽  
Travis L. Rodkey ◽  
Johanna Schaub ◽  
James A. McNew
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Specific Interaction ◽  
Coiled Coil ◽  
Snare Complex ◽  
Juxtamembrane Region

ABSTRACT Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the“ SNARE domain” or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.

scholarly journals Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.

1995 ◽  
Vol 131 (4) ◽  
pp. 913-927 ◽  
Author(s):  
M Lussier ◽  
A M Sdicu ◽  
T Ketela ◽  
H Bussey
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Golgi Localization ◽  
Golgi Compartment ◽  
Membrane Spanning

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

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