| 1. |
- Calado Botelho, Salomé, et al.
(författare)
-
Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria
- 2013
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:7, s. 4792-4798
-
Tidskriftsartikel (refereegranskat)abstract
- Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.
|
|
| 2. |
- Cymer, Florian, et al.
(författare)
-
Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants
- 2015
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:16, s. 10208-10215
-
Tidskriftsartikel (refereegranskat)abstract
- Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.
|
|
| 3. |
- Daniels, Robert, et al.
(författare)
-
Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria
- 2010
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:5, s. 3300-3309
-
Tidskriftsartikel (refereegranskat)abstract
- Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.
|
|
| 4. |
- Galian-Barrueco, Carmen, et al.
(författare)
-
Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity
- 2012
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:20, s. 16399-16409
-
Tidskriftsartikel (refereegranskat)abstract
- Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is marginal: less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.
|
|
| 5. |
- Karamyshev, Andrey L., et al.
(författare)
-
Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains
- 2005
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:49, s. 40489-40493
-
Tidskriftsartikel (refereegranskat)abstract
- Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of similar to 15 residues surrounding a QK*T cross-linking site impairs the interaction between the nascent chain and STT3.
|
|
| 6. |
- Kim, Hyun, et al.
(författare)
-
Membrane topology of the STT3 subunit of the oligosaccharyl transferase complex
- 2005
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:21, s. 20261-20267
-
Tidskriftsartikel (refereegranskat)abstract
- The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N-cyt-C-lum orientation.
|
|
| 7. |
|
|
| 8. |
- Lara, Patricia, et al.
(författare)
-
Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase
- 2019
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:12, s. 4538-4545
-
Tidskriftsartikel (refereegranskat)abstract
- Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.
|
|
| 9. |
|
|
| 10. |
- Lloris-Garcera, Pilar, et al.
(författare)
-
Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
- 2012
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:31, s. 26052-26059
-
Tidskriftsartikel (refereegranskat)abstract
- The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.
|
|
