scholarly journals Tim18p Is a New Component of the Tim54p-Tim22p Translocon in the Mitochondrial Inner Membrane

2000 ◽  
Vol 11 (1) ◽  
pp. 103-116 ◽  
Author(s):  
Oliver Kerscher ◽  
Naresh B. Sepuri ◽  
Robert E. Jensen
Keyword(s):  
Growth Defect ◽  
Inner Membrane ◽  
New Members ◽  
Mitochondrial Inner Membrane ◽  
Native Electrophoresis ◽  
Multiple Copies ◽  
New Gene ◽  
Synthetically Lethal ◽  
Blue Native Electrophoresis ◽  
Blue Native

The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitivetim54-1 mutant. We identified a new gene,TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress thetim54-1 growth defect; thetim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an ∼300-kDa complex after blue native electrophoresis. We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.

scholarly journals The Tim54p–Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane

1997 ◽  
Vol 139 (7) ◽  
pp. 1663-1675 ◽  
Author(s):  
Oliver Kerscher ◽  
Jason Holder ◽  
Maithreyan Srinivasan ◽  
Roxanne S. Leung ◽  
Robert E. Jensen
Keyword(s):  
The Other ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Inner Membrane ◽  
Temperature Sensitive Mutant ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Multiple Copies ◽  
New Protein

We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p–Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p–Tim22p).

High throughput two-dimensional blue-native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes

PROTEOMICS ◽  
2002 ◽  
Vol 2 (8) ◽  
pp. 969 ◽  
Author(s):  
Paul S. Brookes ◽  
Anita Pinner ◽  
Anup Ramachandran ◽  
Lori Coward ◽  
Stephen Barnes ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Proteomics ◽  
Two Dimensional ◽  
Native Electrophoresis ◽  
Blue Native Electrophoresis ◽  
Blue Native

scholarly journals Targeting and assembly of the oxoglutarate carrier: general principles for biogenesis of carrier proteins of the mitochondrial inner membrane

1998 ◽  
Vol 333 (1) ◽  
pp. 151-158 ◽  
Author(s):  
Annamaria PALMISANO ◽  
Vincenzo ZARA ◽  
Angelika HÖNLINGER ◽  
Angelo VOZZA ◽  
Peter J. T. DEKKER ◽  
...  
Keyword(s):  
High Efficiency ◽  
Dependent Manner ◽  
Yeast Mitochondria ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Native Electrophoresis ◽  
Surface Receptor ◽  
Inner Membrane Protein ◽  
Oxoglutarate Carrier

We have studied the targeting and assembly of the 2-oxoglutarate carrier (OGC), an integral inner-membrane protein of mitochondria. The precursor of OGC, synthesized without a cleavable presequence, is transported into mitochondria in an ATP- and membrane potential-dependent manner. Import of the mammalian OGC occurs efficiently into both mammalian and yeast mitochondria. Targeting of OGC reveals a clear dependence on the mitochondrial surface receptor Tom70 (the 70 kDa subunit of the translocase of the outer mitochondrial membrane), whereas a cleavable preprotein depends on Tom20 (the 20 kDa subunit), supporting a model of specificity differences of the receptors and the existence of distinct targeting pathways to mitochondria. The assembly of minute amounts of OGC imported in vitro to the dimeric form can be monitored by blue native electrophoresis of digitonin-lysed mitochondria. The assembly of mammalian OGC and fungal ADP/ATP carrier occurs with high efficiency in both mammalian and yeast mitochondria. These findings indicate a dynamic behaviour of the carrier dimers in the mitochondrial inner membrane and suggest a high conservation of the assembly reactions from mammals to fungi.

Blue native electrophoresis as a tool for detection of PIP-containing protein complexes in the plasma membranes

2013 ◽  
Vol 60 (4) ◽  
pp. 563-570
Author(s):  
T. A. Shevyreva ◽  
M. S. Piotrovskii ◽  
B. V. Belugin ◽  
I. M. Zhestkova ◽  
M. S. Trofimova
Keyword(s):  
Plasma Membranes ◽  
Protein Complexes ◽  
Native Electrophoresis ◽  
Blue Native Electrophoresis ◽  
Blue Native

scholarly journals Tim18p, a New Subunit of the TIM22 Complex That Mediates Insertion of Imported Proteins into the Yeast Mitochondrial Inner Membrane

2000 ◽  
Vol 20 (4) ◽  
pp. 1187-1193 ◽  
Author(s):  
Carla M. Koehler ◽  
Michael P. Murphy ◽  
Nikolaus A. Bally ◽  
Danielle Leuenberger ◽  
Wolfgang Oppliger ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Integral Membrane Protein ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Protein Insertion ◽  
Mitochondrial Inner Membrane ◽  
Precursor Proteins ◽  
Synthetically Lethal

ABSTRACT Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

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