scholarly journals Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins.

1989 ◽  
Vol 109 (6) ◽  
pp. 2603-2616 ◽  
Author(s):  
L Pon ◽  
T Moll ◽  
D Vestweber ◽  
B Marshallsay ◽  
G Schatz
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Buoyant Density ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Membranes ◽  
Outer Membranes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Dependent Protein

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.

scholarly journals Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria.

1993 ◽  
Vol 121 (6) ◽  
pp. 1233-1243 ◽  
Author(s):  
A Mayer ◽  
R Lill ◽  
W Neupert
Keyword(s):  
Outer Membrane ◽  
Protein Transport ◽  
Protein Translocation ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Surface Receptors ◽  
Outer Membranes

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.

scholarly journals Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 414 ◽  
Author(s):  
Justin Nice ◽  
Nataliya Balashova ◽  
Scott Kachlany ◽  
Evan Koufos ◽  
Eric Krueger ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Aggressive Periodontitis ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Type I ◽  
Outer Membranes ◽  
Independent Mechanism ◽  
Bacterial Outer Membrane

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

scholarly journals Role of membrane contact sites in protein import into mitochondria

2015 ◽  
Vol 24 (3) ◽  
pp. 277-297 ◽  
Author(s):  
Susanne E. Horvath ◽  
Heike Rampelt ◽  
Silke Oeljeklaus ◽  
Bettina Warscheid ◽  
Martin van der Laan ◽  
...  
Keyword(s):  
Protein Import ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

scholarly journals Protein Import Channel of the Outer Mitochondrial Membrane: a Highly Stable Tom40-Tom22 Core Structure Differentially Interacts with Preproteins, Small Tom Proteins, and Import Receptors

2001 ◽  
Vol 21 (7) ◽  
pp. 2337-2348 ◽  
Author(s):  
Chris Meisinger ◽  
Michael T. Ryan ◽  
Kerstin Hill ◽  
Kirstin Model ◽  
Joo Hyun Lim ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Stable Complex ◽  
Intermembrane Space ◽  
Import Receptors ◽  
Conductance States ◽  
Coupled Channel

ABSTRACT The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a ∼400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

scholarly journals ORP5 Transfers Phosphatidylserine To Mitochondria And Regulates Mitochondrial Calcium Uptake At Endoplasmic Reticulum - Mitochondria Contact Sites

2019 ◽  
Author(s):  
Leila Rochin ◽  
Cécile Sauvanet ◽  
Eeva Jääskeläinen ◽  
Audrey Houcine ◽  
Amita Arora ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Calcium Uptake ◽  
Vesicular Transport ◽  
Mitochondrial Calcium ◽  
Mitochondrial Membranes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Mitochondrial Calcium Uptake

SUMMARYMitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the two mitochondrial membranes. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at sites of close organelle apposition called membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that ORP5/8 mediate non-vesicular transport of Phosphatidylserine (PS) from the ER to mitochondria in mammalian cells. We also show that ER-mitochondria contacts where ORP5/8 reside are physically and functionally linked to the MIB/MICOS complexes that bridge the mitochondrial membranes, cooperating with them to facilitate PS transfer from the ER to the mitochondria. Finally, we show that ORP5 but not ORP8, additionally regulates import of calcium to mitochondria and consequently cell senescence.

scholarly journals Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane.

1989 ◽  
Vol 109 (2) ◽  
pp. 487-493 ◽  
Author(s):  
S Hwang ◽  
T Jascur ◽  
D Vestweber ◽  
L Pon ◽  
G Schatz
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Protein Translocation ◽  
Membrane Vesicles ◽  
Inner Membrane ◽  
Precursor Proteins ◽  
Membrane Barrier ◽  
Complete Protein ◽  
Membrane Components

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.

scholarly journals The archaeal Sec–dependent protein translocation pathway

2004 ◽  
Vol 359 (1446) ◽  
pp. 919-927 ◽  
Author(s):  
Albert Bolhuis
Keyword(s):  
Protein Import ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Model Systems ◽  
Post Translational Modification ◽  
Sequencing Data ◽  
Archaeal Protein ◽  
Domains Of Life ◽  
Dependent Protein ◽  
Translocation Pathway

Over the past three decades, transport of proteins across cellular membranes has been studied extensively in various model systems. One of the major transport routes, the so–called Sec pathway, is conserved in all domains of life. Very little is known about this pathway in the third domain of life, archaea. The core components of the archaeal, bacterial and eucaryal Sec machinery are similar, although the archaeal components appear more closely related to their eucaryal counterparts. Interestingly, the accessory factors of the translocation machinery are similar to bacterial components, which indicates a unique hybrid nature of the archaeal translocase complex. The mechanism of protein translocation in archaea is completely unknown. Based on genomic sequencing data, the most likely system for archaeal protein translocation is similar to the eucaryal co–translational translocation pathway for protein import into the endoplasmic reticulum, in which a protein is pushed across the translocation channel by the ribosome. However, other models can also be envisaged, such as a bacterial–like system in which a protein is translocated post–translationally with the aid of a motor protein analogous to the bacterial ATPase SecA. This review discusses the different models. Furthermore, an overview is given of some of the other components that may be involved in the protein translocation process, such as those required for protein targeting, folding and post–translational modification.

cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart

1987 ◽  
Vol 65 (2) ◽  
pp. 137-143 ◽  
Author(s):  
James W. Burgess ◽  
Esther W. Yamada
Keyword(s):  
Protein Kinase ◽  
High Speed ◽  
Kinase Activity ◽  
Mitochondrial Protein ◽  
Membrane Vesicles ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Histone H2b ◽  
Outer Membranes ◽  
Dependent Protein

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine – diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.

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