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M T Ryan

Publications and source records attributed to M T Ryan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

Biological Transport↗
PubMed1999

Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane.

The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein.

Adenosine Diphosphate↗
PubMed1999

Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway.

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Amino Acid Sequence↗
PubMed1999

Communicating effectively with regulatory agencies in the licensing process.

Planning preparing, submitting and tracking license and permit applications through the regulatory process is complex and time consuming. A disciplined approach can make the process easier. This paper discusses practical steps to take and some guidelines to follow in making the licensing or permitting process easier.

Communication↗
PubMed1998

Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment].

The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.

Amino Acid Sequence↗
PubMed1998

Rational treatment of panic disorder with antidepressants.

Rational treatment of panic disorders with antidepressants rests on decisions of drug choice, dosage, and duration of treatment. In this paper, we selectively review the author's research with the standard antidepressant, imipramine, in the treatment of panic disorder with agoraphobia as it relates to practical issues. We develop general guidelines for treatment, suggest researchable ways of increasing the net effectiveness of treatment with this class of drugs, and discuss limited generalizations to the extant literature on selective serotonin reuptake inhibitors in panic disorders.

Agoraphobia↗
PubMed1998

Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of approximately 400,000 ( approximately 400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of approximately 100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.

Adenosine Triphosphatases↗
PubMed1998

The preprotein translocase of the mitochondrial outer membrane.

The translocase components of the mitochondrial outer membrane (Tom) are essential for the transfer of preproteins from the cytosol to the mitochondria. At least nine Tom components have now been identified and they fall into two categories; receptor components and components of the General Import Pore (GIP). The receptor components most likely diffuse laterally in the membrane and deposit preproteins atthe GIP where they are then inserted into the mitochondria. This review focuses on these components and their known interactions in the process of preprotein translocation.

Adenosine Triphosphatases↗
PubMed1997

The genes encoding mammalian chaperonin 60 and chaperonin 10 are linked head-to-head and share a bidirectional promoter.

Chaperonins are a class of stress-inducible molecular chaperones involved in protein folding. We report the cloning, sequencing and characterisation of the rat mitochondrial chaperonin 60 and chaperonin 10 genes. The two genes are arranged in a head-to-head configuration and together comprise 14 kb and contain 14 introns. The genes are linked together by a region of approximately 280 bp, which constitutes a bidirectional promoter and includes a common heat-shock element. Insertion of the shared promoter region between two reporter genes is sufficient to drive their expression under both constitutive and heat-shock conditions. The arrangement of the mammalian chaperonin genes suggests the potential to provide the coordinated regulation of their products in a manner that is mechanistically distinct from, yet conceptually similar to, that employed by the bacterial chaperonin (groE) operon.

Animals↗
PubMed1997

The role of molecular chaperones in mitochondrial protein import and folding.

Molecular chaperones play a critical role in many cellular processes. This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein. It also reviews the role of molecular chaperons in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress. Finally, the role of molecular chaperones, in particular to mitochondrial chaperonins, in disease is reviewed. In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones. Thus, studies of protein folding in bacteria such as Escherichia coli have proved to be instructive in understanding the process in the eukaryotic cell. As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses. The regulation of stress genes involved in mitochondrial chaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified.

Animals↗
PubMed1996

Cutaneous sparganosis.

Sparganosis is an infection caused by migrating larvae of the cestode genus Spirometra. There have been approximately 62 cases of sparganosis reported in the United States. Although a subcutaneous mass is the most common manifestation, sparganosis is not well-described in the dermatology literature. We present a case of cutaneous sparganosis in a 52-year-old Filipino American woman. Histologically, the sections showed a granulomatous panniculitis and dermatitis containing a section of a sparganum. A transverse section of an intact sparganum reveals an eosinophilic cuticle, loose stroma, calcareous bodies, and smooth muscle fibers.

Female↗
PubMed1995

Affinity purification, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation.

Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.

Acetylation↗
PubMed1995

Affinity-purification and identification of GrpE homologues from mammalian mitochondria.

We used affinity chromatography on DnaK columns to identify a mitochondrial GrpE homologue from bovine, porcine and rat liver mitochondria. The 24 kDa GrpE homologue bound specifically to the DnaK column and was not eluted with 1 M KCl but readily with 5 mM ATP. Sequence analysis of the bovine homologue (85 residues) revealed 42% positional identity to mitochondrial GrpEp from S. cerevisiae and about 30% identity to the bacterial counterparts. Thus, GrpE homologues from higher and lower eukaryotes are highly conserved.

Amino Acid Sequence↗
PubMed1995

Solution structure of the acetylated and noncleavable mitochondrial targeting signal of rat chaperonin 10.

Chaperonin 10 (Cpn10) is one of only a few mitochondrial matrix proteins synthesized without a cleavable targeting signal. Using a truncated form of Cpn10 and synthetic peptides in mitochondrial import assays, we show that the N-terminal region is both necessary and sufficient for organellar targeting in vitro. To elucidate the structural features of this topogenic signal, peptides representing residues 1-25 of rat Cpn10 were synthesized with and without the naturally occurring N-terminal acetylation. 1H NMR spectroscopy in 20% CF3CH2OH,H2O showed that both peptides assume a stable helix-turn-helix motif and are highly amphiphilic in nature. Chemical shift and coupling constant data revealed that the N-terminal helix is stabilized by N-acetylation, whereas NOE and exchange studies were used to derive a three dimensional structure for the acetylated peptide. These findings are discussed with respect to a recent model predicting that targeting sequences forming a continuous alpha-helix of more than 11 residues cannot adopt a conformation necessary for proteolysis by the matrix located signal peptidases (Hammen, P. K., Gorenstein, D. G., and Weiner, H. (1994) Biochemistry 33, 8610-8617).

Acetylation↗
PubMed1995

Cutaneous angiosarcoma arising in the radiation site of a congenital hemangioma.

We describe a patient in whom angiosarcoma developed at the site of a hemangioma that was treated during infancy with radiation for refractory thrombocytopenia. Our findings, along with those of the 10 reported cases from the world literature, are summarized. One third of angiosarcomas arise in the skin. They most often show one of three clinical patterns. First and most common is occurrence as a bruiselike lesion on the scalp or face of an elderly person. Second in frequency is the Stewart-Treves syndrome. Third and least common is angiosarcoma developing as a sequela of previous radiation therapy. The prognosis in general is poor, with a mean survival length of 24 months and a 5-year survival rate of 10%. Effective treatment relies on early diagnosis and wide-margin surgical excision.

Fatal Outcome↗
PubMed1995

Role of chaperones in the biogenesis and maintenance of the mitochondrion.

All cells depend on correctly folded proteins for optimal function. A central question in cellular biology is how such folded structures are formed and maintained, a process that is now recognized to rely heavily on a group of proteins called molecular chaperones. Molecular chaperones constitute distinct families of proteins that are ubiquitous and highly conserved from bacteria to humans. They appear to bind nonnative conformations of most, if not all, proteins, thereby preventing their aggregation and subsequent inactivation. The chaperones not only protect newly synthesized proteins during transport and folding, but also serve to maintain the cell in a healthy state during exposure to a multitude of stress conditions. Accordingly, chaperones are expressed constitutively, but their synthesis is further enhanced during stress conditions. Detailed insights into the role of molecular chaperones have come from studies of mitochondrial protein biogenesis, a process in which chaperones act as unfoldases, pulling devices, and foldases. In this review we summarize these developments and further discuss the potential role of chaperones in mitochondrial DNA metabolism and human mitochondrial disease states.

Animals↗