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Are porins on the inner or on the outer membrane of mitochondria?

Are porins on the inner or on the outer membrane of mitochondria?


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I've looked at multiple resources and they are saying different things.


This is not my field, but The Transporter Classification Database would appear to be a reliable source and states that:

The best characterized members of the MPP family are the voltage-dependent anion-selective channel (VDAC) porins in the mitochondrial outer membrane.

Searching through the Protein Data Bank one can find a crystal structure for human voltage-dependent anion channel 1 and the associated paper also states that it is in the outer mitochondrial membrane, so you would imagine that they should know.


Intermembrane space

The intermembrane space is the region between the inner membrane and the outer membrane of a mitochondrion or a chloroplast. Its main function is nucleotide phosphorylation . Channel proteins called porins in the outer membrane allow free movement of ions and small molecules into the intermembrane space.

As electrons move down the proteins in the electron transport chain, the electrons lose energy to bring H+ ions from the mitochondrial matrix into the intermembrane space.

Intermembrane Space
The intermembrane space (short IMS) of the mitochondria is the region that lies between the outer and inner membrane of the mitochondria. This is the place where the oxidative phosphorylation takes place [14].
Mitochondrial Matrix .

is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space.

- The space between the outer and inner membrane in a mitochondria.
Lysosome - A membrane-bound organelle found in eukaryotic cells. Contain acids and enzymes that degrade unwanted molecules.
Matrix - The space inside the inner membrane of mitochondria.

of the mitochondria
C. Inner membrane of the mitochondria
D. Matrix of the mitochondria .

The ellipsoid-shaped chloroplast is enclosed in a double membrane and the area between the two layers that make up the membrane is called the

. The outer layer of the double membrane is much more permeable than the inner layer, which features a number of embedded membrane transport proteins.

The interior of the two membranes is called the matrix, the space in between the two membranes is called the

and the folds created by the inner membrane are called cristae. Mitochondria also contain their own DNA which encodes some of the enzymes that are used inside the mitochondria.

from the mitochondrial matrix.
293
98 .

The proton gradient develops between the

The mitochondrion consists of outer and inner membranes, an

(space in between the membranes), the cristae (infoldings of the inner membrane), and the matrix (space within the inner membrane). The outer membrane contains several porins that form channels where certain molecules can freely diffuse.

The energy released as the electrons are passed from carrier to carrier moves hydrogen ions (protons) across the membrane, from the mitochondrial matrix to the

, creating a concentration gradient of protons.

After cyanide poisoning, the electron transport chain can no longer pump electrons into the

would increase, and ATP synthesis would stop.
B
C .

mitochondria mitochondrian outer membrane inner membrane cristae

The components of oxidative phosphorylation-the electron transport chain and ATP synthase-are embedded within the inner mitochondrial membrane. Protons (+) are pumped from the matrix into the fluid-filled space between the two membranes, also called the "

The space between the outer and inner membranes is called the

. Inside the inner membrane are flat, pancake-like structures called thylakoids. The thylakoids form stacks called grana.


Functions of Mitochondria

Mitochondria are responsible for many major activities inside a cell:

  • It is the main site for ATP synthesis and therefore it is called a powerhouse of the cell (Oxidative Phosphorylation). The cells like muscle cells, liver, indicate the higher rate of ATP utilization in these areas. In mature RBCs, mitochondria are absent in order to create more space for haemoglobin for oxygen binding.
  • The inner membrane of mitochondria has proteins (F0-F1 particles) which are- involved in electron transport and ATP synthesis.

  • Mitochondria has a vital role in uptake and release of Ca2+ which maintains the concentration of calcium in the cytoplasm of the cells.
  • Mitochondria also play a very essential role in the process of Apoptosis in mammalian cells. The proteins of BCL2 family regulates the release of cytochrome c from the inner and outer membrane of mitochondria which, once in cytoplasm, causes activation of other apoptotic signals, finally causing cell death.
  • In adipose tissues, mitochondria release heat via electron transport chain.

Biocenter of the University of Würzburg, Würzburg, Germany

Biocenter of the University of Würzburg, Würzburg, Germany

Abstract

The mitochondrial outer membrane contains a channel that is responsible for the passage of hydrophilic metabolites across the membrane. The channel-forming protein known for many organisms, called mitochondrial porin or VDAC (voltage-dependent anion-selective channel), has a length of about 280 amino acids. The genomes of eukaryotic organisms contain several porin isoforms of not well-defined function. The primary structure of mitochondrial or eukaryotic porins is not particularly hydrophobic and secondary structure predictions suggest that a β-barrel cylinder typical for the secondary structure of bacterial porins also forms the mitochondrial channel. Kinases involved in mitochondrial metabolism such as hexokinase or glycerokinase bind to porin and play an important role in compartment formation in mitochondria. Mitochondrial porins are voltage-gated and switch into ion-permeable substates at voltages higher than 20 to 30 mV. The open channel has a small preference for anion over cations of the same aqueous mobility. The closed states are cation selective and impermeable for ATP and ADP. Mitochondrial porins seem to be involved in apoptosis and the release of cytochrome c. There exists emerging evidence that the cytoplasmic membrane of eukaryotic cells also contains porins of the eukaryotic porin family with a role in cellular metabolism that is not yet understood.


What are Mitochondria, and Why Do They Matter?

Every living thing is comprised of cells , which are the building blocks of life. Inside of these cells are a series of organell es – sub-compartment-like entities that perform various but specific functions . Different cells throughout your body notably have different shapes and sizes, and thus, different purposes. Similar c ells (ones that do the same job ) form body tissue s , such as muscle, skin, or bone tissue. Groups of different types of cells make up the organs in your body, such as your heart, liver, or lungs. Collectively, all organs work together as a system to keep you alive and healthy ( Science NetLinks ) .

What are mitochondria?

“The powerhouse of the cell” that’s how most people think of and remember the mitochondri on (plural: mitochondria) from their high school biology class. The y are organell e s that function like microscopic , yet super complex factories , produc ing energy and dispos ing of waste that is detrimental to the body. Mitochondria are critical for cell survival , and ATP (energy) that your mitrochondria help produce is vital for metabolic processes and keeping you healthy .

The Structure of Mitochondria

Mitochondria are designed to maximize their productivity. They contain two membranes – the outer membrane functions like a skin, and the inner membrane makes it possible for more reactions to occur. This means that your cells can get more work done. Outer membrane: This is a phospholipid bilayer that includes protein-based structures called porins, which enable molecules (ions, ATP, ADP, etc.) to cross.

Inner membrane: This membrane is highly complex and i t i s where most ATP is created. It includes all the complexes of the electron transport system , the ATP synthetase complex , and transport proteins. The inner membrane does not have porins like the outer membrane, so it is impermeable to most molecules.

Cristae: These are the folds of the inner membrane, which increase the surface area and the space available for chemical reactions to occur. This is also where the electron transport chain and enzymes are located. The number of cristae in the mitochondria correlates with the given cell’s demand for ATP. For example, heart muscle cells contain up to three times more cristae than other cells due to the greater need for ATP ( Biology Dictionary ).

Matrix: This is the space within the inner membrane where the citric acid cycle , or Krebs cycle, takes place. This is an important part of cellular respiration and ATP production. Mitochondrial DNA is also housed here.

How the Mighty Mitochondria Work

Mitochondria are found in the cells of animals, humans, plants and fungi. While the y are primarily known for converting energy from food to energy for biological processes, mitochondria are deeply involved in several other activities that enable cells to function efficiently t o help you keep your body healthy. Here are the five key roles that mitochondria play in cellular health, and what can happen when these functions are disturbed:

  1. Production of ATP Mitochondria produce 90% of the energy our body needs to function by convert ing chemical energy from nutrients to ATP . During cellular respiration, another chemical called NADH is produced , which is then used by enzymes to generate ATP in the form of chemical bonds. The production of ATP is essential to help the body function properly. Without energy, your cells and your body suffer. Dysfunction from lack of ATP can contribute to a variety of health concerns, including but not limited to : Alzheimer’s , Parkinson’s, L ou Gehrig’s disease , diabetes , cancer , and several types of mitochondrial diseas e .
  2. Calcium Homeostasis — Thi s is the flow of calcium in and out of a cell’s mitochondria . This process is an important part of metabolic regulation and killing off unhealthy cells. When mitochondrial calcium homeostasis is compromised, different pathological conditions can occur, depending on the cell type involved (NCBI).
  3. Cell Migration This refers to the orchestrated movement of cells to specific locations in response to chemical signals. Regulation of cell migration can help terminate unhealthy cells and speed up wound-healing conversely, i f this process is not well-managed, there may be serious health consequences, including but not limited to: tumor formation and can cer formation, vascular disease, tumor formation and metastasis. – This is essentially programmed cell death which involves maintaining the health of the body by eliminating old cells, unnecessary cells, and unhealthy cells. Without appropriate apoptosis (either too little or too much), there is a greater risk of experiencing health conditions, i ncluding neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer (NCBI) .
  4. Innate Immunity — This refers to nonspecific defense mechanisms – such as skin, chemicals in the blood, and cells within your immune system that attack foreign cells — that basically come to the rescue immediately or within hours of an antigen’s appearance in the body . In addition to regulating antiviral signaling, mitochondria also contribute to innate immune activation following cellular damage and stress ( NCBI ) .

To recap, ATP is energy currency for your cells, and your mitochondria are th e primary sources for producing more of it to help keep you alive and healthy . These organelles are also responsible for surveilling and eliminating the growth of unhealthy cells. If any deficiency in your mitochondria are present, then you are more likely to experience health issue s .


Results

Expression of VDAC in E. coli

To investigate whether the E. coli Bam complex can deal with a mitochondrial β-barrel protein, we genetically fused VDAC from N. crassa to the signal sequence of the bacterial porin PhoE to allow for transport across the inner membrane. In addition to the full-length protein, two VDAC variants were initially constructed to investigate whether the β-signal, which is required for the assembly of β-barrel proteins into the mitochondrial OM, is also needed for their assembly into the bacterial OM. Deletion of two amino acid residues from the C terminus does not affect the β-signal identified by Kutik et al. (2008) and positions a Phe residue at the C terminus like in the bacterial OMP signature sequence ( fig. 1). Deletion of five residues does affect the β-signal and eliminates the Phe residue ( fig. 1). The constructs generated are under control of the phoE promoter, which allows for high-level expression when the cells are grown under Pi limitation. Because they are located on a medium-copy plasmid, low-level expression was anticipated under Pi-replete conditions.

Comparison of the bacterial and mitochondrial β-barrel-OMP assembly signals and the C termini of the VDAC variants used in this study. One-letter code for amino acids is used. X, any amino acid φ, hydrophobic residue π, polar residue n, 1–28 residues. The mitochondrial β-signal is bordered by thick lines.

Comparison of the bacterial and mitochondrial β-barrel-OMP assembly signals and the C termini of the VDAC variants used in this study. One-letter code for amino acids is used. X, any amino acid φ, hydrophobic residue π, polar residue n, 1–28 residues. The mitochondrial β-signal is bordered by thick lines.

Cells of the wild-type E. coli strain MC4100 containing the relevant plasmids were grown in Pi-replete L-broth, and whole-cell lysates were analyzed by SDS-PAGE and western blotting. Immunodecoration of the blots revealed that full-length VDAC was expressed under these conditions ( fig. 2A). The specificity of the VDAC antiserum was confirmed by the absence of a signal in the strain carrying the empty vector ( fig. 2A). After ultrasonic disruption of the E. coli cells, VDAC fractionated with the membranes like the endogenous porins OmpF and OmpC, whereas the cytoplasmic chaperone SecB was recovered in the supernatant ( fig. 2A). Deletion of two residues from the C terminus did not influence protein levels, and like the full-length protein, this mutant form was also recovered in the membrane fraction ( fig. 2A). However, the mutant protein lacking five C-terminal residues was not detected under these conditions, possibly because this variant was not assembled into the OM and thus was proteolytically degraded. Similarly, VDAC was not detected when expressed without a signal sequence ( fig. 2A). To verify the proteolytic degradation of the mutant protein lacking five C-terminal residues, the plasmid encoding this construct was introduced into strain JW0157, which lacks the major periplasmic protease DegP, and its isogenic parental strain BW25113. Because the mutant protein was detected only in the degP mutant ( fig. 2B), it appears to be a substrate for the periplasmic protease in the wild-type strain.

Mitochondrial VDAC from Neurospora crassa is localized to the OM in Escherichia coli. (A) Whole-cell lysates, cell envelopes, and soluble fractions (supernatant) were isolated from MC4100 cells containing plasmids encoding VDAC (full length), C-terminal truncated variants lacking five (Δ5 C-term.) or two (Δ2 C-term.) amino acids, VDAC without signal sequence (Δ signal) or the empty vector. The fractions were analyzed by SDS-PAGE and western blotting using antisera directed against VDAC, E. coli porins (OmpC/F), or the cytoplasmic protein SecB. (B) Whole cells of E. coli strain BW25113 and its degP mutant derivative JW0157, both containing plasmids encoding either full-length VDAC or mutant VDAC lacking five C-terminal residues, were analyzed by SDS-PAGE followed by western blotting using antisera directed against VDAC or OmpA. (C) Inner membranes and OMs from MC4100 cells expressing full-length VDAC were separated by sucrose density gradient centrifugation. Fractions were analyzed by SDS-PAGE and western blotting using antisera directed against VDAC or E. coli porins. NADH-oxidase activity was assessed as an inner-membrane marker. Signals or activities for each protein in each fraction were normalized to the amount detected in the peak fraction. (D) Mitochondria isolated from N. crassa (left) and suspensions of cell envelopes from E. coli strain MC4100 expressing N. crassa VDAC (right) were either left intact (input), digested with the indicated concentrations of proteinase K (PK), or extracted with 6 M urea and separated into an extractable (supernatant) and a membrane-embedded (pellet) fraction. Samples were subjected to SDS-PAGE, followed by either staining with Coomassie Brilliant Blue to detect the OmpC, OmpF, and OmpA proteins or western blotting using antibodies against VDAC and Tom40. Tom40, mitochondrial OMP with a protease-accessible loop OmpA, E. coli OMP with protease-accessible periplasmic extension OmpC and OmpF, protease-protected OMPs.

Mitochondrial VDAC from Neurospora crassa is localized to the OM in Escherichia coli. (A) Whole-cell lysates, cell envelopes, and soluble fractions (supernatant) were isolated from MC4100 cells containing plasmids encoding VDAC (full length), C-terminal truncated variants lacking five (Δ5 C-term.) or two (Δ2 C-term.) amino acids, VDAC without signal sequence (Δ signal) or the empty vector. The fractions were analyzed by SDS-PAGE and western blotting using antisera directed against VDAC, E. coli porins (OmpC/F), or the cytoplasmic protein SecB. (B) Whole cells of E. coli strain BW25113 and its degP mutant derivative JW0157, both containing plasmids encoding either full-length VDAC or mutant VDAC lacking five C-terminal residues, were analyzed by SDS-PAGE followed by western blotting using antisera directed against VDAC or OmpA. (C) Inner membranes and OMs from MC4100 cells expressing full-length VDAC were separated by sucrose density gradient centrifugation. Fractions were analyzed by SDS-PAGE and western blotting using antisera directed against VDAC or E. coli porins. NADH-oxidase activity was assessed as an inner-membrane marker. Signals or activities for each protein in each fraction were normalized to the amount detected in the peak fraction. (D) Mitochondria isolated from N. crassa (left) and suspensions of cell envelopes from E. coli strain MC4100 expressing N. crassa VDAC (right) were either left intact (input), digested with the indicated concentrations of proteinase K (PK), or extracted with 6 M urea and separated into an extractable (supernatant) and a membrane-embedded (pellet) fraction. Samples were subjected to SDS-PAGE, followed by either staining with Coomassie Brilliant Blue to detect the OmpC, OmpF, and OmpA proteins or western blotting using antibodies against VDAC and Tom40. Tom40, mitochondrial OMP with a protease-accessible loop OmpA, E. coli OMP with protease-accessible periplasmic extension OmpC and OmpF, protease-protected OMPs.

Assembly of VDAC in the Bacterial OM

We wished to determine in which membrane VDAC was located. To separate inner membranes and OMs, we applied sucrose density gradient centrifugations. VDAC fractionated with the marker OM proteins OmpF and OmpC and not with the inner-membrane marker NADH-oxidase ( fig. 2C). Next, we monitored the integration of VDAC into the OM in protease-digestion experiments. In its native environment, the mitochondrial OM, the protein is resistant to proteinase K, whereas Tom40, which has a large loop exposed to the surface of the mitochondria, is degraded ( fig. 2D). Also in the bacterial OM, VDAC was largely resistant to proteinase K, like the endogenous porins OmpC and OmpF and unlike OmpA, which has a large periplasm-exposed domain and was degraded under these conditions ( fig. 2D). Furthermore, akin the bacterial porins, VDAC could not be extracted from the bacterial membranes with urea ( fig. 2D), confirming that it was integrally inserted into the OM.

If VDAC is indeed assembled into the bacterial OM, it might be detectable at the bacterial cell surface with specific antibodies. The low-level background expression in uninduced MC4100 cells was insufficient for detection of VDAC by immunofluorescence microscopy. To increase the expression levels, the plasmid encoding VDAC was introduced into E. coli strain CE1224, which does not produce endogenous porins. Growing the transformed cells under Pi limitation resulted in drastic overproduction of VDAC, even to such an extent that a small fraction of unprocessed precursor proteins was detected ( fig. 3A). Using immunofluorescence microscopy, the protein was detected at the bacterial cell surface, like it was at the surface of isolated mitochondria ( fig. 3B). The specificity of the signal observed was confirmed by its absence in bacteria carrying the empty vector. Also, the mutant protein lacking two C-terminal amino acid residues was detected at the bacterial cell surface, but not the one lacking five amino acids ( fig. 3B), although it was abundantly produced under these conditions ( fig. 3C). Collectively, these results indicate that VDAC assembles into the bacterial OM and that assembly is dependent on the β-signal.

Detection of VDAC on the bacterial cell surface using immunofluorescence microscopy. (A) Steady-state levels of full-length VDAC in different Escherichia coli strains used in this study. Escherichia coli strain CE1224 was grown under Pi deprivation, whereas strains CE1265 and MC4100 were exponentially grown in L-broth. All strains contained plasmid pJPNcPor1 encoding full-length VDAC under the control of the phoE promoter. Equal amounts of cells were harvested. Cell envelopes were isolated, serially diluted and analyzed by SDS-PAGE and western blotting using an antiserum against VDAC. Accumulation of unprocessed precursor (P) containing the signal sequence is observed in the fully induced CE1224 cells. (B) Isolated mitochondria from Neurospora crassa or E. coli CE1224 cells expressing full-length or C-terminally truncated VDAC variants, or carrying an empty vector, grown overnight under Pi deprivation, were fixed and immunostained with an antiserum against VDAC followed by a fluorophore-conjugated secondary antibody. Images were acquired in fluorescence (FL) or bright field (BF) channels. (C) Comparison of the expression levels of full-length VDAC and the mutant protein lacking five C-terminal residues (Δ5 C-term.) in strain CE1224 grown under Pi limitation. Equal amounts of cells were harvested and analyzed by SDS-PAGE and western blotting with antiserum against VDAC. P, precursor and M, mature form.

Detection of VDAC on the bacterial cell surface using immunofluorescence microscopy. (A) Steady-state levels of full-length VDAC in different Escherichia coli strains used in this study. Escherichia coli strain CE1224 was grown under Pi deprivation, whereas strains CE1265 and MC4100 were exponentially grown in L-broth. All strains contained plasmid pJPNcPor1 encoding full-length VDAC under the control of the phoE promoter. Equal amounts of cells were harvested. Cell envelopes were isolated, serially diluted and analyzed by SDS-PAGE and western blotting using an antiserum against VDAC. Accumulation of unprocessed precursor (P) containing the signal sequence is observed in the fully induced CE1224 cells. (B) Isolated mitochondria from Neurospora crassa or E. coli CE1224 cells expressing full-length or C-terminally truncated VDAC variants, or carrying an empty vector, grown overnight under Pi deprivation, were fixed and immunostained with an antiserum against VDAC followed by a fluorophore-conjugated secondary antibody. Images were acquired in fluorescence (FL) or bright field (BF) channels. (C) Comparison of the expression levels of full-length VDAC and the mutant protein lacking five C-terminal residues (Δ5 C-term.) in strain CE1224 grown under Pi limitation. Equal amounts of cells were harvested and analyzed by SDS-PAGE and western blotting with antiserum against VDAC. P, precursor and M, mature form.

Pore Formation by VDAC in the E. coli OM

VDAC is the main pathway by which metabolites cross the outer mitochondrial membrane. Thus, if VDAC is correctly assembled into the bacterial OM, it should form large pores. To test this possibility, the plasmid encoding VDAC was introduced into E. coli strain CE1265, which does not produce endogenous porins. Due to a mutation in the regulatory gene phoR, this strain expresses the pho regulon constitutively but not to the same extent as does a fully induced strain like CE1224 ( fig. 3A). The formation of pores in the OM was determined in an antibiotic sensitivity test in which the zone of growth inhibition around a disc containing the antibiotic tested is a measure for the diffusion of the antibiotic through the pores. When the bacteria produced VDAC, they were more sensitive to all antibiotics tested ( table 1), indicating that VDAC forms pores in the bacterial OM. To substantiate this conclusion, the cleavage of nitrocefin by periplasmic β-lactamase was measured in intact cells, a process in which the permeation of this chromogenic antibiotic through the OM is rate limiting. The synthesis of VDAC resulted in a drastically increased rate of nitrocefin cleavage in intact cells ( fig. 4A) consistent with the formation of aqueous pores in the OM. In both assays, expression of phoE from the same vector resulted in a more moderate increase in antibiotic uptake ( table 1 and fig. 4A) consistent with the observation that VDAC has a considerably larger pore size than the E. coli porins ( Freitag et al. 1982). Collectively, similar to its role in mitochondria, the integration of VDAC into the bacterial OM promotes the flux of solutes across this membrane.

Expression of VDAC in Escherichia coli Cells Increases their Susceptibility to Antibiotics.

Plasmid Growth Inhibition Zone (mm)
Ery (25) Amp (5) Tet (2) Rif (0.3) Vnc (5) Ceph (5)
Vector 4.0 4.3 3.7 1.0 1.7 2.0
PhoE 6.0 5.7 6.3 2.3 1.0 4.0
VDAC 15.7 16.7 10.7 14.0 12.3 6.3
Plasmid Growth Inhibition Zone (mm)
Ery (25) Amp (5) Tet (2) Rif (0.3) Vnc (5) Ceph (5)
Vector 4.0 4.3 3.7 1.0 1.7 2.0
PhoE 6.0 5.7 6.3 2.3 1.0 4.0
VDAC 15.7 16.7 10.7 14.0 12.3 6.3

N OTE .—Porin-less E. coli strain CE1265 was transformed with an empty vector, a plasmid encoding PhoE, or a plasmid encoding VDAC. The susceptibility of the transformed cells to antibiotics was assessed on plates by measuring the growth inhibition zone around filter discs soaked with the antibiotics at the concentration indicated in brackets (in mg/ml). Mean values of growth inhibition zone (in mm from the rim of the filter discs) from three experiments are shown. Ery, erythromycin Amp, ampicillin Tet, tetracycline Rif, rifamycin Vnc, vancomycin and Ceph, cephaloridine.

Expression of VDAC in Escherichia coli Cells Increases their Susceptibility to Antibiotics.

Plasmid Growth Inhibition Zone (mm)
Ery (25) Amp (5) Tet (2) Rif (0.3) Vnc (5) Ceph (5)
Vector 4.0 4.3 3.7 1.0 1.7 2.0
PhoE 6.0 5.7 6.3 2.3 1.0 4.0
VDAC 15.7 16.7 10.7 14.0 12.3 6.3
Plasmid Growth Inhibition Zone (mm)
Ery (25) Amp (5) Tet (2) Rif (0.3) Vnc (5) Ceph (5)
Vector 4.0 4.3 3.7 1.0 1.7 2.0
PhoE 6.0 5.7 6.3 2.3 1.0 4.0
VDAC 15.7 16.7 10.7 14.0 12.3 6.3

N OTE .—Porin-less E. coli strain CE1265 was transformed with an empty vector, a plasmid encoding PhoE, or a plasmid encoding VDAC. The susceptibility of the transformed cells to antibiotics was assessed on plates by measuring the growth inhibition zone around filter discs soaked with the antibiotics at the concentration indicated in brackets (in mg/ml). Mean values of growth inhibition zone (in mm from the rim of the filter discs) from three experiments are shown. Ery, erythromycin Amp, ampicillin Tet, tetracycline Rif, rifamycin Vnc, vancomycin and Ceph, cephaloridine.

VDAC forms pores in the bacterial OM. (A) Cells from strain CE1265 containing pBR322 encoding β-lactamase were used. The cells were additionally transformed with the empty vector or a plasmid encoding either PhoE or the indicated VDAC variants and incubated with nitrocefin. Turnover of the substrate was determined by spectrophotometry. (B) Whole-cell lysates from cells used in nitrocefin-uptake experiments were analyzed by SDS-PAGE and western blotting using antibodies against the indicated proteins.

VDAC forms pores in the bacterial OM. (A) Cells from strain CE1265 containing pBR322 encoding β-lactamase were used. The cells were additionally transformed with the empty vector or a plasmid encoding either PhoE or the indicated VDAC variants and incubated with nitrocefin. Turnover of the substrate was determined by spectrophotometry. (B) Whole-cell lysates from cells used in nitrocefin-uptake experiments were analyzed by SDS-PAGE and western blotting using antibodies against the indicated proteins.

VDAC Assembly in the E. coli OM is Dependent on the β-Signal

In the nitrocefin-uptake assay, expression of the mutant VDAC lacking two residues from the C terminus promoted cleavage of the antibiotic at efficiencies similar to those obtained upon expression of the full-length protein ( fig. 4A), suggesting that this mutation does not affect assembly and pore formation. In contrast, nitrocefin cleavage was much less promoted by expression of the variant lacking five residues ( fig. 4A). The latter variant could be detected in the constitutive strain CE1265 although clearly less than the full-length protein ( fig. 4B). The protein was largely degraded when the cell envelope fraction of these cells was treated with proteinase K and it could be extracted from these membranes with urea, whereas the full-length protein produced in this strain was protease resistant and not extractable from the membranes with urea ( fig. 5A). Thus, it appears that a large fraction of the detected mutant protein was not inserted into the OM these polypeptides only fractionate with the membranes, presumably because they form dense aggregates that are pelleted with the membranes during the centrifugation procedure ( Tommassen 1986). Nevertheless, a small portion of the polypeptides was resistant to proteinase K and was not extracted with urea ( fig. 5A) these polypeptides are most likely correctly assembled into the bacterial OM as they facilitated nitrocefin degradation in intact cells to some extent ( fig. 4A) consistent with the formation of functional pores.

VDAC assembly into the Escherichia coli OM depends on an intact β-signal. (A) The majority of VDAC molecules lacking the five C-terminal residues is not properly integrated into the OM. Cell envelopes from E. coli strain CE1265 expressing either the full-length VDAC (upper part) or the mutant VDAC (lower part) were treated with proteinase K (PK) or extracted with urea as described in the legend to figure 2D and analyzed by SDS-PAGE and either staining with Coomassie Brilliant Blue to visualize OmpA or western blotting to detect the VDAC variant. (B) Cell envelopes of E. coli strain MC4100 expressing either full-length VDAC or the mutant protein lacking four C-terminal residues were extracted with urea as described in the legend to figure 2D and analyzed by SDS-PAGE and western blotting using antiserum against VDAC.

VDAC assembly into the Escherichia coli OM depends on an intact β-signal. (A) The majority of VDAC molecules lacking the five C-terminal residues is not properly integrated into the OM. Cell envelopes from E. coli strain CE1265 expressing either the full-length VDAC (upper part) or the mutant VDAC (lower part) were treated with proteinase K (PK) or extracted with urea as described in the legend to figure 2D and analyzed by SDS-PAGE and either staining with Coomassie Brilliant Blue to visualize OmpA or western blotting to detect the VDAC variant. (B) Cell envelopes of E. coli strain MC4100 expressing either full-length VDAC or the mutant protein lacking four C-terminal residues were extracted with urea as described in the legend to figure 2D and analyzed by SDS-PAGE and western blotting using antiserum against VDAC.

The results presented so far indicated that the mutant protein lacking five C-terminal residues is only very inefficiently incorporated into the E. coli OM, whereas the mutant protein lacking two residues, which still retains an intact β-signal and presents a Phe residue at the C terminus, is assembled as efficiently as is the wild-type VDAC. To investigate whether the presence of a C-terminal Phe could compensate for the disruption of the β-signal, an additional mutant was constructed, which lacks four amino acid residues from the C terminus where it presents a Phe ( fig. 1). In contrast to the mutant protein lacking five C-terminal residues ( fig. 2A), the protein lacking four residues was detected when the construct was expressed in the wild-type E. coli strain. However, when the membrane fraction of this strain was extracted with urea, the mutant protein was partially solubilized, whereas the wild-type protein was insoluble as before ( fig 5B). Therefore, in spite of the presence of a Phe at the C terminus, the mutant protein lacking four C-terminal residues is inefficiently assembled into the E. coli OM. Together, our results are consistent with the notion that an intact β-signal is needed for efficient insertion of VDAC into the bacterial OM but is not absolutely essential for this process.

VDAC Assembly Is Dependent on BamA

To determine whether assembly of VDAC into the OM of E. coli depends on the Bam complex, we made use of a temperature-sensitive (ts) bamA mutant strain ( Doerrler and Raetz 2005). This strain fails to grow at 44 °C, but even at a permissive temperature, it shows an assembly defect of OMPs as evidenced by decreased levels of these proteins. These reduced levels probably result from degradation of misassembled OMPs by periplasmic proteases, such as DegP. The plasmid encoding VDAC was introduced into this mutant strain and into an isogenic strain carrying a wild-type bamA gene, and the bacteria were grown at 32 °C under Pi-limiting conditions to induce VDAC synthesis. The bamA(ts) mutation did not affect growth under these conditions ( fig. 6A). Like the amounts of correctly assembled endogenous porins and OmpA, the amount of membrane-inserted VDAC was drastically reduced in the bamA(ts) mutant strain ( fig. 6B). In contrast, levels of the periplasmic enzyme alkaline phosphatase (PhoA), the inner–membrane-located enzyme leader peptidase, and the cytoplasmic chaperone SecB in the cell lysates were unaffected ( fig. 6B). Thus, BamA is required for the efficient assembly of VDAC into the bacterial OM.

BamA is required for membrane insertion of VDAC in Escherichia coli. (A) Escherichia coli cells containing either a ts allele or wild-type bamA were grown overnight in L-broth at 30 °C. Cells were resuspended at time 0 in Pi-poor medium (LPi) or the same medium supplemented with 660 μM K2HPO4 (HPi) and growth at 32 °C was determined by measuring the optical density at 660 nm. (B) Pi-starved cells containing either the ts bamA allele or the wild-type allele grown in LPi or HPi were harvested after 6 h (arrow in panel A). Cell envelopes were isolated and extracted with 6 M urea to determine the amount of membrane-inserted VDAC and E. coli OMPs. Whole-cell lysates were analyzed to determine the effect of the bamA ts mutation on other proteins. Samples were analyzed by SDS-PAGE, followed either by staining with Coomassie Brilliant Blue to visualize PhoE, OmpC, OmpF, and OmpA or by western blotting using antisera against the following controls: PhoA, periplasmic alkaline phosphatase, which is expressed upon Pi limitation leader peptidase, an inner-membrane protein SecB, a cytoplasmic protein.

BamA is required for membrane insertion of VDAC in Escherichia coli. (A) Escherichia coli cells containing either a ts allele or wild-type bamA were grown overnight in L-broth at 30 °C. Cells were resuspended at time 0 in Pi-poor medium (LPi) or the same medium supplemented with 660 μM K2HPO4 (HPi) and growth at 32 °C was determined by measuring the optical density at 660 nm. (B) Pi-starved cells containing either the ts bamA allele or the wild-type allele grown in LPi or HPi were harvested after 6 h (arrow in panel A). Cell envelopes were isolated and extracted with 6 M urea to determine the amount of membrane-inserted VDAC and E. coli OMPs. Whole-cell lysates were analyzed to determine the effect of the bamA ts mutation on other proteins. Samples were analyzed by SDS-PAGE, followed either by staining with Coomassie Brilliant Blue to visualize PhoE, OmpC, OmpF, and OmpA or by western blotting using antisera against the following controls: PhoA, periplasmic alkaline phosphatase, which is expressed upon Pi limitation leader peptidase, an inner-membrane protein SecB, a cytoplasmic protein.


Mitochondria are double-membrane and rods shaped organelles found in the cells of both animals and plants and are responsible for various metabolic functions within the cells. Mitochondria play a vital role in providing the necessary biological energy for the cell through enzymatic oxidation of the substrates of the Krebs&rsquos cycle. The mitochondrion is made up of the outer membrane, inner membrane, intermembrane space, and matrix.

The outer membrane consists of the same amounts of proteins and phospholipids as well as a large number of porins which are specialized proteins. The outer membrane is permeable to ions, nutrients, and energy molecules such as ADP and ATP (Abdrakhimova et al, 2011). The inner membrane of the mitochondrion is complex and consists of a number of folds known as the cristae which are important since it increases the surface area within the organelle. The increased surface area within the organelle coupled with the presence of various proteins within the inner membrane is crucial in aiding various enzymatic reactions including the production of ATP.

However, unlike the outer membrane, the inner membrane is strictly permeable to ATP, Oxygen, and the movement of metabolites across the membrane (Jones, 2013). The space between the outer and the inner membranes is referred to as the intermembrane space and its composition is similar to that of the cytoplasm. The matrix which is the fourth component of the mitochondrion is made up of a complex mixture of enzymes and proteins which are important in the synthesis of the ribosome, ATP, mitochondrial DNA, and tRNAs (Miles et al, 2014).

Succinate dehydrogenase is one of the enzyme complex bound to the inner membrane of the mitochondrion. Succinate dehydrogenase plays a vital role in Krebs&rsquos cycle since it catalyzes the oxidation of Succinate to fumarate within the inner membrane (Gorbacheva et al, 2013).


How do proteins enter mitochondria?

If the inner membrane is so impermeable, how do proteins enter?

The outer membrane of the mitochondria contains the protein "porin". This forms an aqueous channel through which proteins up to 10,000 daltons can pass and go into the intermembrane space. Indeed, the small molecules actually equilibrate between the outer membrane and the cytosol. However, most proteins cannot get into the matrix unless they pass through the inner membrane. This membrane contains cardiolipin which renders it virtually impermeable. This requires transport mechanisms across the membrane that are more organized and regulated. A very simple view of the process is diagrammed in this cartoon.

This figure is taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing, N.Y. 1994, Third Edition

Transport across the mitochondrial membranes requires the concerted action of a number of translocation machineries. The machinery in the outer membrane is called the Tom complex (Translocator outer membrane) and that for the inner membrane is called the Tim complex (Translocator Inner Membrane). Proteins that have to go all the way to the matrix have an NH2 cleavable signal sequence (see the above cartoon).

Most proteins must be uncoiled or stretched out to go through the translocators. This involves ATP binding and is monitored and stabilized by a chaperone proteins, including hsp70. Thus, before the protein can go through Tom complex, it must become "translocation competent".

Transport through the outer membrane: characteristics of Tom complex.

Not surprisingly, the TOM complex will include import receptors that initially recognize the signal peptide or a signal sequence (these include Tom20, Tom22, and Tom70). Different proteins use different receptors. In the above cartoon, the receptor is represented as a blue oval in which the signal peptide is inserted. The receptors then bring the protein to the region containing the translocator proteins. This is actually a complex of proteins.
It is called the General Import Pore (GIP) and it facilitates the translocation of the presequence of the protein across the outer membrane. (the GIP is made of Tom40, Tom5, Tom 6, and Tom7). Tom40 appears to be the core element of the pore and forms oligomers. It traverses the membrane as a series of 14 anti-parallel beta strands which form a beta barrel. It also interacts with polypeptide chains passing through the pore. All of the other Tom components in GIP are anchored to the outer membrane by helical transmembrane segments (hydrophobic anchors).

Recent study of Tom40: Rapaport, D and Neupert W, Biogenesis of Tom40, Core Component of the TOM complex of mitochondria. J Cell Biol 146 321-332, 1999. Study looked at how Tom40 entered outer membrane and became a part of the GIP. The study reported that:

  • First, as with many mitochondrial proteins, Tom40 requires cytosolic chaperones to prepare it for entry. In the case of this protein, becoming "translocation competent" requires ATP and a partially folded state (the latter is mediated by the cytosolic chaperone (hsp70).
  • Second, when it is "competent", it interacts with the surface receptor, Tom20. There is no cleavable signal peptide however, the experiments showing the requirement for partial folding suggests targeting information is found in discontinuous sites brought together in the folded domain.
  • Final insertion is into preexisting Tom complexes. This requires an intact N terminus.
  • Dimerization occurs after entry into the membrane.

Characteristics of Tim Complexes

Mitochondrial proteins destined for the matrix often have a cleavable signal peptide on the protein which must be recognized before it will be admitted through the mitochondrial translocator. These proteins with "amino terminal signals" (your text), or "preproteins" or "presequences" (current literature) usually interact with Tom20 first. Then, they have to get through the outer membrane. To do that, they are transferred to the GIP complex: First, they interact with Tom22 and Tom5 which ushers them to the pore formed by Tom40. They then enter the matrix using the pore complex made of Tim23 and Tim17 which are in the inner membrane. Also, very important, their entry is dependent on membrane potential. This is set up by the electron transport complexes. Recall that hydrogen ions are being pumped into the intermembrane space creating a charge gradient that is more negative on the matrix site. This membrane potential actually helps pulls the protein into the Tim23-Tim17 channels. The protein then enters the matrix where the cleavable preprotein is clipped off by a protease, MPP. mt- hsp 70 in the matrix works with Tim44 to complete the full transfer to the matrix. mthsp70 and Tim 44 actually "pull" the protein into the matrix by a process that requires ATP. It also requires the membrane potential set up by the electron transport chain.

Some mitochondrial proteins destined for the inner membrane have a cleavable presequence followed by one or more hydrophobic membrane-spanning segments that function as stop-transfer sequences in the IM or, serve to insert the polypeptide into the IM after it gets in the matrix. These are like the Type I membrane proteins described in the unit on the rough endoplasmic reticulum.

However, other proteins do not have a cleavable targeting signal (Types II and III). Mitochondrial proteins that have an internal signal sequence (examples include a number of proteins in the inner membrane) generally interact with Tom70 as the receptor. Then, after they transit the outer membrane via the GIP complex, they enter the special Tim pathway. This may involve interactions with small Tim's of the intermembrane space and Tim22-Tim54 of the inner membrane itself.

Those proteins that do not have a cleavable targeting signal sequence often have signals with the following characteristics: They are often a stretch of positively charged amino acids (sometimes adjacent to a membrane spanning hydrophobic region). Sometimes these form loops that face the matrix. Recall the "positive inside rule" has positively charged amino acids concentrated at the cytosolic side for proteins being inserted into the rough endoplasmic reticulum. These mitochondrial proteins tend to follow this rule, only the matrix becomes the site where the positive charges are most numerous.

Examples from the literature:

Davis, AJ, Ryan, KR, and Jensen, RE Tim23p contains separate and distinct signals for targeting to mitochondria and insertion in to the inner membrane. Molecular Biology of the Cell 9: 2577-2593 (1999).

  • Tim23 is one of the inner membrane translocator proteins. It does not have an amino-terminal presequence. Targeting information is found within the mature protein.
  • Tim23 has 4 transmembrane segments and two positively charged loops facing the matrix. What is needed for signalling an import?
  • Replaced positively charged amino acids in one or both loops with alanine residues.
  • At least one of these loops is required for insertion into the inner membrane.
  • The signal for targeting to mitochondria is found in at least two of the hydrophobic transmembrane segments.

Kurz, M, Martin, H, Rassow J, Pfanner, N, and Ryan, MT. Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway. Molecular Biology of the Cell 10: 2461-2474 (1999). Compared the route and binding of three Tim proteins

  • Tim54 carries a amino terminal, noncleaved translocation sequence that is positively charged. However, it prefers to use Tom70 as its receptor instead of Tom20. After moving through the GIP, it uses its positively charged amino terminal sequence to enter the matrix. It required chaperones and ATP to get to the matrix.
  • Tim22 is a hydrophobic protein that uses Tom20 for targeting to the OM. Then it follows the Tim route for carrier proteins, like Tim23. It does not require hsp70 or ATP for entry.
  • Small Tims are normally found in the intermembrane space and are not membrane proteins. They used Tom20 for their receptor and transfer to the GIP complex. However, when Tom20 was destroyed by trypsin, leaving only Tom5, the small Tims were able to enter.

The above cartoon from your text shows more ways that proteins can be inserted into inner and outer membranes, once they are recognized by the receptors. As shown by proteins in the literature examples above, the mitochondria uses both positively charged signals as well as membrane spanning hydrophobic sequences to translocate and then reach their final destination. As in the above examples, there can be multiple signal and insertion sites. However, the distribution of the charged amino acids helps orient the protein so that the positive charges are in the matrix. This is how the cytochromes in the respiratory chain or the elementary particles are inserted by mitochondrial actions.

This figure is taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing, N.Y. 1994, Third Edition

The following figure is from another text by Lodish et al, Molecular Cell Biology. It shows the entire sequence of events required to take a protein into the matrix.

  • Step 1: Protein unfolds as it binds to hsp70 chaperone. Red positive area indicates targeting sequence. Chaperone binding is ATP dependent.
  • Step 2: Targeting sequence binds to receptor (usually Tom20)
  • Step 3. Receptor ushers protein to site of translocator. Other Tom proteins involved, but Tom40 is the core of the translocator channel.
  • Step 4: Protein is translocated stimulated by the membrane potential. Electron transport complexes on inner membrane have pumped H+ across to the intermembrane space, leaving the matrix more negative. This attracts the protein (the signal is positively charged). Protein moves through Tim translocators. Tim 44 and hsp70 in the matrix continue to guide and pull the protein through the pore. An ATP requiring process.
  • Step 5. another chaperone (called a chaperonin), hsp60 causes the folding of the the protein into its tertiary sequence. Also an ATP requiring process.
  • Step 6. Presequence is cleaved in the matrix.

What happens if an import protein is defective?

Studies of yeast have helped us learn about the receptor and translocation machinery contains a complex of proteins that work together to allow entry. In yeast, these have been named the MOMX. series, where the number designates the protein number. An important protein in the recognition of the signal peptide and its binding to the receptor is called "MOM19". MOM 19 works with MOM 72 to recognize and bind the proteins. Then MOM22 helps the protein to pass from the receptor binding site to the insertion point at the outer membrane. The importance of MOM19 can be proved by adding antibodies to MOM19 and blocking import.

In a recent paper by Harkness et al (J Cell Biology 124: 637-648, 1995), they created mutant yeast cells that included a defective gene for MOM19.

They also included a drug resistant marker so they could selectively grow cells with the mutant gene (in the presence of the drug, p=fluorophenyl alanine, or fpa). So, the longer the cells grow in the drug, the more drug-resistant mutant cells will be found. The above electron micrographs are from their paper (cited above). They show the result of the absent MOM19 protein. What is absent in the cells grown for 16 or 32 h in the drug?

When they did the assays for the proteins, what proteins were actually missing? Tests showed that there was a dramatic decrease in most of the respiratory chain (electron transport chain) including cytochromes a/a3, and b. However, cytochrome C was unaffected. This suggests that another protein must control its import.


Differential Centrifugation

Differential centrifugation is the most common method of fractionating cell. Fractionation is separation of different organelles within a cell. It is a classical procedure used to isolate different particles by stepwise successive centrifugations at increasing RCF's (Relative Centrifugal Forces).
Centrifugation separates particles in a suspension based on differences in size, shape and density that together define their sedimentation coefficient. The tube containing the suspension of particles is rotated at a high speed, which exerts a centrifugal force directed from the center of the rotor towards the bottom of the tube. Centrifugal Force 'G' is more commonly expressed as the Relative Centrifugal Force (RCF) or g value in multiples of the earth's gravitational field 'g'.

Relative centrifugal force or g value is calculated using the following formula:

RCF =Relative Centrifugal Force,
r = radius in centimeter,
Q = revolutions per minute.

Thus depending on the radius of centrifuge being used the Q value will vary for different centrifuge to obtain the same g value. When doing differential centrifugation, density of the liquid in which the centrifugation is carried out should be uniform and its density must be far lower than that of the particles to be separated. The viscosity of the particles should also be very low. As a consequence, the rate of particle sedimentation depends on its size and the applied g force. Differential centrifugation gives a crude resolution of sub cellular fraction. This centrifugation is usually carried out using fixed angle rotor.


Mitochondria: Structure and Functions

Mitochondria are known as the powerhouses of the cell. They are organelles that act like a digestive system that takes in nutrients, breaks them down, and creates energy-rich molecules for the cell. The biochemical processes of the cell are known as cellular respiration. Many of the reactions involved in cellular respiration happen in the mitochondria. They are the working organelles that keep the cell full of energy.

There are small organelles floating free throughout the cell. Some cells have several thousand mitochondria while others have none. Muscle cells need a lot of energy so they have loads of mitochondria. Neurons (cells that transmit nerve impulses) don’t need as many. If a cell feels it is not getting enough energy to survive, more mitochondria can be created. Sometimes it can grow larger or combine with other mitochondria. It all depends on the needs of the cell.


Metabolons and Supramolecular Enzyme Assemblies

Kendrick B. Turner , . Scott A. Walper , in Methods in Enzymology , 2019

2.2.1.2 Cultivation conditions

Cultivation conditions as described in Section 2.1.1.2 can be followed until the induction of expression (steps 1–4). The subsequent protocols are based on the protein–protein packaging strategy described by Alves et al. that employs a SpyTagged-porin protein (OmpA-ST) and a SpyCatcher-labeled phosphotriesterase enzyme (PTE-SC) ( Alves et al., 2018 Alves, Turner, Medintz, et al., 2016 Zakeri et al., 2012 ). The methods below are based on a two vector expression system in which the anchor protein (OmpA-ST) is under the control of an arabinose-inducible promoter, and the enzyme (PTE-SC) is regulated using a T7 promoter. Expression of the T7 polymerase is itself regulated by a lactose-inducible promoter.

After the culture reaches an OD600 = 0.8 add l -arabinose (0.02%–1% final concentration v/v) to induce expression of the PTE-SC construct under the control of the arabinose inducible promoter.

Incubate an additional 2 h at 37°C.

Induce expression of the OmpA-ST anchor by adding the lactose analog Isopropyl β- d -1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM.


Watch the video: Porin 2008: Porin za životno djelo - Zvonko Špišić (July 2022).


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