scholarly journals Bacterial tail anchors can target to the mitochondrial outer membrane

2017 ◽  
Author(s):  
Güleycan Lutfullahoğlu Bal ◽  
Abdurrahman Keskin ◽  
Ayşe Bengisu Seferoğlu ◽  
Cory D. Dunn
Keyword(s):  
Outer Membrane ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Mitochondrial Outer Membrane ◽  
Bacterial Proteins ◽  
Rate Limiting Step ◽  
Protein Entry ◽  
Rate Limiting

ABSTRACTDuring the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was refashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol. Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM. Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

scholarly journals Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

1996 ◽  
Vol 16 (8) ◽  
pp. 4035-4042 ◽  
Author(s):  
D A Court ◽  
F E Nargang ◽  
H Steiner ◽  
R S Hodges ◽  
W Neupert ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Protein Translocation ◽  
Specific Binding ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Terminal Domain ◽  
Tom Complex

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.

scholarly journals LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

2019 ◽  
Author(s):  
Florian Lindner ◽  
Bailey Milne-Davies ◽  
Katja Langenfeld ◽  
Andreas Diepold
Keyword(s):  
Temporal Resolution ◽  
Type Iii Secretion ◽  
Protein Delivery ◽  
Protein Translocation ◽  
Eukaryotic Cell ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Reporter Protein ◽  
Effector Secretion ◽  
Control Protein

AbstractMany bacteria employ a type III secretion system (T3SS), also called injectisome, to translocate proteins into eukaryotic host cells through a hollow extracellular needle. The system can efficiently transport heterologous cargo, which makes it a uniquely suited tool for the translocation of proteins into eukaryotic cells. However, the injectisome indiscriminately injects proteins into any adjoining eukaryotic cell, and this lack of target specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to control protein secretion and translocation into eukaryotic cells by light. By combining optogenetic interaction switches with the dynamic cytosolic T3SS component SctQ, the cytosolic availability of SctQ and in consequence T3SS-dependent effector secretion can be regulated by external light. The resulting system, which we call LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the application of the system for light-regulated translocation of a heterologous reporter protein into cultured eukaryotic cells. LITESEC-T3SS represents a new method to achieve unparalleled spatial and temporal resolution for the controlled protein translocation into eukaryotic host cells.

scholarly journals Mutant huntingtin does not cross the mitochondrial outer membrane

2020 ◽  
Vol 29 (17) ◽  
pp. 2962-2975
Author(s):  
James Hamilton ◽  
Tatiana Brustovetsky ◽  
Rajesh Khanna ◽  
Nickolay Brustovetsky
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Alkali Treatment ◽  
Mitochondrial Protein ◽  
Postmortem Brain ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mutant Huntingtin ◽  
Mitochondrial Protein Import ◽  
Brain Tissues

Abstract Mutant huntingtin (mHTT) is associated with mitochondria, but the exact mitochondrial location of mHTT has not been definitively established. Recently, it was reported that mHTT is present in the intermembrane space and inhibits mitochondrial protein import by interacting with TIM23, a major component of mitochondrial protein import machinery, but evidence for functional ramifications were not provided. We assessed mHTT location using synaptic and nonsynaptic mitochondria isolated from brains of YAC128 mice and subjected to alkali treatment or limited trypsin digestion. Mitochondria were purified either with discontinuous Percoll gradient or with anti-TOM22-conjugated iron microbeads. We also used mitochondria isolated from postmortem brain tissues of unaffected individuals and HD patients. Our results demonstrate that mHTT is located on the cytosolic side of the mitochondrial outer membrane (MOM) but does not cross it. This refutes the hypothesis that mHTT may interact with TIM23 and inhibit mitochondrial protein import. The levels of expression of nuclear-encoded, TIM23-transported mitochondrial proteins ACO2, TUFM, IDH3A, CLPP and mitochondrially encoded and synthesized protein mtCO1 were similar in mitochondria from YAC128 mice and their wild-type littermates as well as in mitochondria from postmortem brain tissues of unaffected individuals and HD patients, supporting the lack of deficit in mitochondrial protein import. Regardless of purification technique, mitochondria from YAC128 and WT mice had similar respiratory activities and mitochondrial membrane potentials. Thus, our data argue against mHTT crossing the MOM and entering into the mitochondrial intermembrane space, making it highly unlikely that mHTT interacts with TIM23 and inhibits protein import in intact mitochondria.

scholarly journals Assembly of outer-membrane proteins in bacteria and mitochondria

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2587-2596 ◽  
Author(s):  
Jan Tommassen
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Basic Mechanism ◽  
Eukaryotic Cell ◽  
Mitochondrial Outer Membrane ◽  
Central Component ◽  
Cell Organelles ◽  
C Terminus

The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic α-helices, integral outer-membrane proteins (OMPs) form β-barrels. Similar β-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these β-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of β-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial β-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

scholarly journals Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

2016 ◽  
Vol 113 (31) ◽  
pp. E4467-E4475 ◽  
Author(s):  
Sandro Käser ◽  
Silke Oeljeklaus ◽  
Jiří Týč ◽  
Sue Vaughan ◽  
Bettina Warscheid ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
In Vitro Assays ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Genomes ◽  
Dual Function ◽  
Complex Assembly

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

scholarly journals Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

1989 ◽  
Vol 109 (4) ◽  
pp. 1421-1428 ◽  
Author(s):  
J Rassow ◽  
B Guiard ◽  
U Wienhues ◽  
V Herzog ◽  
F U Hartl ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Outer Membrane ◽  
Dihydrofolate Reductase ◽  
Protein Translocation ◽  
Close Contact ◽  
Membrane Surface ◽  
Mitochondrial Protein ◽  
Contact Sites ◽  
Precursor Proteins ◽  
The Matrix

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Sign in / Sign up

Export Citation Format

Share Document