Two isoforms of PSAP/MTCH1 share two proapoptotic domains and multiple internal signals for import into the mitochondrial outer membrane

2007 ◽  
Vol 293 (4) ◽  
pp. C1347-C1361 ◽  
Author(s):  
Violeta Lamarca ◽  
Antonio Sanz-Clemente ◽  
Rosaura Pérez-Pé ◽  
María José Martínez-Lorenzo ◽  
Nabil Halaihel ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Transmembrane Domain ◽  
Cultured Cells ◽  
Mitochondrial Protein ◽  
Transmembrane Domains ◽  
Presenilin 1 ◽  
Mitochondrial Outer Membrane ◽  
Rat Tissues ◽  
Mitochondrial Carrier ◽  
Amino Terminal

Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed.

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 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 Membrane Topology of the NixA Nickel Transporter ofHelicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III

2000 ◽  
Vol 182 (6) ◽  
pp. 1722-1730 ◽  
Author(s):  
John F. Fulkerson ◽  
Harry L. T. Mobley
Keyword(s):  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Membrane Topology ◽  
Structural Elements ◽  
Transmembrane Helices ◽  
Translational Initiation ◽  
Amino Terminal ◽  
Localization Signal ◽  
Critical Residues

ABSTRACT NixA, the high-affinity cytoplasmic membrane nickel transport protein of Helicobacter pylori, imports Ni2+into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with β-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni2+ transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni2+transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.

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 Pore formation by dimeric Bak and Bax: an unusual pore?

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160218 ◽  
Author(s):  
Rachel T. Uren ◽  
Sweta Iyer ◽  
Ruth M. Kluck
Keyword(s):  
Outer Membrane ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Apoptotic Cell ◽  
Hydrophobic Surface ◽  
Mitochondrial Pathway ◽  
Membrane Curvature ◽  
Basic Unit ◽  
Mitochondrial Outer Membrane ◽  
Membrane Pores

Apoptotic cell death via the mitochondrial pathway occurs in all vertebrate cells and requires the formation of pores in the mitochondrial outer membrane. Two Bcl-2 protein family members, Bak and Bax, form these pores during apoptosis, and how they do so has been investigated for the last two decades. Many of the conformation changes that occur during their transition to pore-forming proteins have now been delineated. Notably, biochemical, biophysical and structural studies indicate that symmetric homodimers are the basic unit of pore formation. Each dimer contains an extended hydrophobic surface that lies on the outer membrane, and is anchored at either end by a transmembrane domain. Membrane-remodelling events such as positive membrane curvature have been reported to accompany apoptotic pore formation, suggesting Bak and Bax form lipidic pores rather than proteinaceous pores. However, it remains unclear how symmetric dimers assemble to porate the membrane. Here, we review how clusters of dimers and their lipid-mediated interactions provide a molecular explanation for the heterogeneous assemblies of Bak and Bax observed during apoptosis. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane

1997 ◽  
Vol 323 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Fiona FRASER ◽  
Clark G. CORSTORPHINE ◽  
Victor A. ZAMMIT
Keyword(s):  
Outer Membrane ◽  
Carnitine Palmitoyltransferase ◽  
Transmembrane Domains ◽  
Proteinase K ◽  
Mitochondrial Outer Membrane ◽  
Malonyl Coa ◽  
Cytosolic Side ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Peptide Antibodies

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.

scholarly journals Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

1998 ◽  
Vol 18 (6) ◽  
pp. 3173-3181 ◽  
Author(s):  
Frank E. Nargang ◽  
Doron Rapaport ◽  
R. Gary Ritzel ◽  
Walter Neupert ◽  
Roland Lill
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Wild Type ◽  
Binding Studies ◽  
Amino Terminal ◽  
Precursor Proteins ◽  
Mutant Strains ◽  
Cytosolic Domain ◽  
Charged Residues ◽  
Positively Charged

ABSTRACT TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing atom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutanttom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

scholarly journals Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site.

1988 ◽  
Vol 107 (6) ◽  
pp. 2483-2490 ◽  
Author(s):  
R Pfaller ◽  
H F Steger ◽  
J Rassow ◽  
N Pfanner ◽  
W Neupert
Keyword(s):  
Outer Membrane ◽  
Insertion Site ◽  
Mitochondrial Protein ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Protein Uptake ◽  
Receptor Sites ◽  
Precursor Proteins ◽  
Multiple Receptor

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.

scholarly journals Procaspase-9 is attached to the mitochondrial outer membrane in the early stages of apoptosis

2007 ◽  
Vol 12 (4) ◽  
Author(s):  
Irina Milisav ◽  
Dušan Šuput
Keyword(s):  
Outer Membrane ◽  
Outer Surface ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Rat Hepatocytes ◽  
Mitochondrial Outer Membrane ◽  
Cell Type ◽  
Caspase 9 ◽  
Mitochondrial Protein Import ◽  
Cellular Location

AbstractProcaspase-9 is the zymogen form of one of the apoptosis initiators, caspase-9. Its cellular location may differ depending on the cell type; it is found throughout the cytosol, although some of it may be associated with the mitochondria. Procaspase-9 relocates from the cytosol to the mitochondria shortly after the triggering of apoptosis in rat hepatocytes. We investigated whether the mitochondrial protein import machineries import procaspase-9. The combined results of protein import analyses, mitochondrial fractionation and protease treatments of intact and swollen mitochondria imply that procaspase-9 attaches to the outer surface of the mitochondrial outer membrane.

Sign in / Sign up

Export Citation Format

Share Document