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Theory of Intelligent Design, the best explanation of Origins » Molecular biology of the cell » Metabolism » The Transport of Proteins into Mitochondria

The Transport of Proteins into Mitochondria

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The Transport of Proteins into Mitochondria

Although mitochondria  have their own genetic systems, they produce only a small proportion of their own proteins. Instead, the two organelles import most of their proteins from the cytosol. Proteins are transported in an unfolded state across both outer and inner membranes simultaneously into the matrix space. Both ATP hydrolysis and a membrane potential across the inner membrane drive translocation into mitochondria. Chaperone proteins of the cytosolic hsp70 family maintain the precursor proteins in an unfolded state, and a second set of hsp70 proteins in the matrix space or stroma pulls the polypeptide chain into the organelle. Only proteins that contain a specific signal sequence are translocated. The signal sequence can either be located at the N-terminus and cleaved off after import or be internal and retained. Transport into the inner membrane sometimes uses a second, hydrophobic signal sequence that is unmasked when the first signal sequence is removed.

Proteomic studies have revealed that mitochondria contain more than 1,000 different proteins, more than 99% of which are encoded by nuclear DNA. Thus, these proteins are synthesized as precursors on cytosolic ribosomes, specifically targeted to mitochondria and sorted into one of the four mitochondrial subcompartments. Various protein translocation machineries of the inner and outer mitochondrial membranes accomplish the complicated task of selective protein sorting into and across lipid bilayers. 3

Mitochondria are double-membrane-enclosed organelles. They specialize in ATP synthesis, using energy derived from electron transport and oxidative phosphorylation in mitochondria and from photosynthesis in chloroplasts . Although both organelles contain their own DNA, ribosomes, and other components required for protein synthesis, most of their proteins are encoded in the cell nucleus and imported from the cytosol.

If the endosymbiosis theory were true, would the proteins not keep being encoded and produced inside mitochondria ?

Each imported protein must reach the particular organelle subcompartment in which it functions. There are different subcompartments in mitochondria : the internal matrix space and the intermembrane space, which is continuous with the cristae space. These compartments are formed by the two concentric mitochondrial membranes: the inner membrane, which encloses the matrix space and forms extensive invaginations called cristae, and the outer membrane, which is in contact with the cytosol. Protein complexes provide boundaries at the junctions where the cristae invaginate and divide the inner membrane into two domains: one inner membrane domain surrounds the cristae space, and the otherdomain abuts the outer membrane. Chloroplasts also have an outer and inner membrane, which enclose an intermembrane space, and the stroma, which is the chloroplast equivalent of the mitochondrial matrix space . They have an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. The thylakoid membrane derives from the inner membrane during plastid development and is pinched off to become discontinuous with it. Each of the subcompartments in mitochondria and chloroplasts contains a distinct set of proteins. New mitochondria and chloroplasts are produced by the growth of preexisting organelles, followed by fission . The growth depends mainly on theimport of proteins from the cytosol. The imported proteins must be transported across a number of membranes in succession and end up in the appropriate place. The process of protein movement across membranes is calledprotein translocation. This section explains how it occurs.

Translocation into Mitochondria Depends on Signal Sequences and Protein Translocators

Proteins imported into mitochondria are usually taken up from the cytosol within seconds or minutes of their release from ribosomes. Thus, in contrast to protein translocation into the ER, which often takes place simultaneously with translation by a ribosome docked on the rough ER membrane , mitochondrial proteins are first fully synthesized as mitochondrial precursor proteins in the cytosol and then translocated into mitochondria by a post-translational mechanism. One or more signal sequences direct all mitochondrial precursor proteins to their appropriate mitochondrial subcompartment. Many proteins entering the matrix space contain a signal sequence at their N-terminus that a signal peptidase rapidly removes after import. Other imported proteins, including all outer membrane and many inner membrane and intermembrane space proteins, have internal signal sequences that are not removed. The signal sequences are both necessary and sufficient for the import and correct localization of the proteins: when genetic engineering techniques are used to link these signals to a cytosolic protein, the signals direct the protein to the correct mitochondrial subcompartment.

Question: had this signalling not have to be fully developed and functioning right from the beginning ?

The signal sequences that direct precursor proteins into the mitochondrial matrix space are best understood. They all form an amphiphilic α helix, in which positively charged residues cluster on one side of the helix, while uncharged hydrophobic residues cluster on the opposite side. Specific receptor proteins that initiate protein translocation recognize this configuration rather than the precise amino acid sequence of the signal sequence

A signal sequence for mitochondrial protein import. Cytochrome oxidase is a large multiprotein complex located in the inner mitochondrial membrane, where it functions as the terminal enzyme in the electron-transport chain. (A) The first 18 amino acids of the precursor to subunit IV of this enzyme serve as a signal sequence for import of the subunit into the mitochondrion. (B) When the signal sequence is folded as an α helix, the positively charged amino acids (red) are clustered on one face of the helix, while the nonpolar ones (green) are clustered primarily on the opposite face. Uncharged polar amino acids are shaded orange; nitrogen atoms on the side chains of Arg and Gln are colored blue. Signal sequences that direct proteins into the matrix space always have the potential to form such an amphiphilic α helix, which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an import receptor (gray), as determined by nuclear magnetic resonance. The amphiphilic α helix binds with its hydrophobic face to a hydrophobic groove in the receptor

Multisubunit protein complexes that function as protein translocators mediate protein movement across mitochondrial membranes. The TOM complex transfers proteins across the outer membrane, and two TIM complexes (TIM23 and TIM22) transfer proteins across the inner membrane

These complexes contain some components that act as receptors for mitochondrial precursor proteins, and other components that form the translocation channels. The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins.

This is another reason why the endosymbiosis theory makes no sense. The mechanism of synthesis of all proteins INSIDE the bacteria would change, and a new mechanism where the proteins are synthesised in the nucleus would have to emerge. And so the import mechanism with all related proteins and signalling mechanisms. Not a easy task.

It initially transports their signal sequences into the intermembrane space and helps to insert transmembrane proteins into the outer membrane. β-barrel proteins, which are particularly abundant in the outer membrane, are then passed on to an additional translocator, the SAM complex, which helps them to fold properly in the outer membrane. The TIM23 complex transports some soluble proteins into the matrix space and helps to insert transmembrane proteins into the inner membrane. The TIM22 complex mediates the insertion of a subclass of inner membrane proteins, including the transporter that moves ADP, ATP, and phosphate in and out of mitochondria.

This strongly suggests that the whole mechanism, and so the proteins, had to emerge all at once, and be fully operational right from the start. How did the TIM23 complex " learn " how to transport the soluble proteins into the matrix space, and help to insert transmembrane proteins into the inner membrane ? trial and error ?

Yet another protein translocator in the inner mitochondrial membrane, the OXA complex, mediates the insertion of those inner membrane proteins that are synthesized within mitochondria. It also helps to insert some imported inner membrane proteins that are initially transported into the matrix space by the other complexes

Only 13 proteins necessary for a mitochondrion are actually coded in mitochondrial DNA. The vast majority of proteins destined for the mitochondria are encoded in the nucleus and synthesized in the cytoplasm. These are tagged by an N-terminal signal sequence. Following transport through the cytosol from the nucleus, the signal sequence is recognized by a receptor protein in the transporter outer membrane (TOM) complex. 1

This reinforces the evidence that the endosymbiosis theory is false.

Thats what precisely was to be expected: some wishy washy pseudo scientific speculation about " proto " mitochondrion, and a primitive setup of protein translocases. The problem with this line of reasoning is always, its baseless speculation , where words like " might be ", " it has been suggested ", " would have ", " demonstrates the feasability " are nothing else than assertions without a shred of evidence, just in order to provide a framework, where the evolutionary paradigm might fit. Thats not science. Thats pseudo science at its best.


Last edited by Admin on Mon Aug 10, 2015 5:00 pm; edited 8 times in total

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Protein Transport (Mitochondrial) -- Movie Narrative

Most organelle proteins are synthesized in the cytoplasm from nuclear encoded mRNAs. These proteins must be imported into the organelle. Special sequences, called signal sequences, target the protein to its proper organelle. Organelles contain protein translocator complexes that are required for this transport.

Key players in this process are protein, a signal sequence, chaperonins, ATP, protein translocator complexes, and signal peptidase.

In order for organelle proteins being able to arrive at their destination inside the mitochondria, following is required :

all the machinery to synthesize mRNA's
Cytoplasm and the container of it ( the cell membrane )
signal sequences
protein translocator complexes required for the transport
signal peptidase
and the organelle ( the mitochondrion ) into which the protein is transported

if any of it is missing, nothing goes. That is a irreducible , interlocked and interdependent system, which indicates that all the organelles and machinery had to emerge simultaneously. A separate independent stepwise arise is not possible.

Proteins destined for transport into an organelle, such as a mitochondrion or chloroplast, contain a signal sequence. This sequence acts as a targeting mechanism to ensure the protein is delivered to the proper organelle.

Most signal-relay stations we know about were intelligently designed. Signal without recognition is meaningless.  Communication implies a signalling convention (a “coming together” or agreement in advance) that a given signal means or represents something: e.g., that S-O-S means “Send Help!”   The transmitter and receiver can be made of non-sentient materials, but the functional purpose of the system always comes from a mind.  The mind uses the material substances to perform an algorithm that is not itself a product of the materials or the blind forces acting on them.  Signal sequences may be composed of mindless matter, but they are marks of a mind behind the intelligent design.

In addition, chaperonin proteins aid in the import process. They become associated with a protein while it is still in the cytoplasm. This association require energy from ATP. Chaperonins aid in unfolding the protein, so that it can travel through the organelle membrane.

Question : Had this process not have to be fully functioning right from the beginning, otherwise the proteins would not be able to enter the mitochondria ?

Here we see two chaperonins bound to the protein that will enter the mitochondrion.

Protein translocator complexes are embedded in the mitochondrial membrane. These are multi-protein complexes required for protein import. The protein being transfered first attaches to the complex on the cytosolic side. The protein then moves into the mitochondrion.

Question: Had these protein complexes not have to be fully functioning and existing right from the start, otherwise the process would not be possible to happen ?

As it enters the organelle it is again bound by a chaperonin to prevent premature folding. Once the protein has fully entered the mitochondrion, the first chaperonin is released and another class of chaperonins bind.

Then a complex called the signal peptidase removes the signal sequence.

This seems to be a highly organized and orchestrated process that had to be pre-programmed right from the beginning, and is best explained through the invention and implementation of a creator.

Lastly, the protein is folded into its final shape, and is ready to perform its proper function in the organelle.

The folding is also a highly complex process, and must be just right.

Last edited by Admin on Mon Aug 10, 2015 10:50 am; edited 2 times in total

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Most mitochondrial proteins are synthesized by ribosomes in the cytoplasm, or cytosol, of the cell.

Proteins destined for the mitochondria are identified as such by markers known as signal sequences.

Specialized proteins called chaperones bind to the protein to prevent premature folding and aid in transport to the mitochondria.

Chaperones use the energy from ATP hydrolosis to keep the precursor protein unfolded.

The protein is then guided to the mitochondrial outer membrane.

Special receptors embedded in the outer membrane recognize the protein's signal sequence.

The protein is then transported through pores in the mitochondrial inner and outer membranes. These pores are associated with the receptors and together are known as a protein translocator complex.

Once inside the matrix space, the protein is again bound by a chaperone to prevent folding.

The first chaperone then leaves as new chaperonins attach. These new chaperonins will aid in folding the protein into its final shape.

Soon after entering the matrix, an enzyme known as signal peptidase detects the protein's signal sequence.

Signal peptidase then cleaves the signal sequence from the protein.

The chaperonins finish folding the protein into its final shape.

Once folded into its final shape, the protein is now ready to preform its proper function in the organelle.

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Mitochondrial Precursor Proteins Are Imported as Unfolded Polypeptide Chains

Mitochondrial precursor proteins do not fold into their native structures after they are synthesized; instead, they remain unfolded in the cytosol through interactions with other proteins. Some of these interacting proteins are general chaperone proteins of the hsp70 family , whereas others are dedicated to mitochondrial precursor proteins and bind directly to their signal sequences. All the interacting proteins help to prevent the precursor proteins from aggregating or folding up spontaneously before they engage with the TOM complex in the outer mitochondrial membrane. As a first step in the import process, the import receptors of the TOM complex bind the signal sequence of the mitochondrial precursor protein. The interacting proteins are then stripped off, and the unfolded polypeptide chain is fed—signal sequence first—into the translocation channel. In principle, a protein could reach the mitochondrial matrix space by either crossing the two membranes all at once or crossing one at a time.
Although the TOM and TIM complexes usually work together to translocate precursor proteins across both membranes at the same time, they can work independently. In isolated outer membranes, for example, the TOM complex can translocate the signal sequence of precursor proteins across the membrane. Similarly, if the outer membrane is experimentally disrupted in isolated mitochondria, the exposed TIM23 complex can efficiently import precursor proteins into the matrix space.

ATP Hydrolysis and a Membrane Potential Drive Protein Import Into the Matrix Space

Directional transport requires energy, which in most biological systems is supplied by ATP hydrolysis. ATP hydrolysis fuels mitochondrial protein import at two discrete sites, one outside the mitochondria and one in the matrix space. In addition, protein import requires another energy source, which is the membrane potential across the inner mitochondrial membrane
The first requirement for energy occurs at the initial stage of the translocation process, when the unfolded precursor protein, associated with chaperone proteins, interacts with the import receptors of the TOM complex. As discussed in Chapter 6, the binding and release of newly synthesized polypeptides from the chaperone proteins requires ATP hydrolysis.
Once the signal sequence has passed through the TOM complex and is bound to a TIM complex, further translocation through the TIM translocation channel requires the membrane potential, which is the electrical component of the electrochemical H+ gradient across the inner membrane

Pumping of H+ from the matrix space to the intermembrane space, driven by electron transport processes in the inner membrane, maintains the electrochemical gradient. The energy in the electrochemical H+ gradient across the inner membrane therefore not only powers most of the cell’s ATP synthesis, but it also drives the translocation of the positively charged signal sequences through the TIM complexes by electrophoresis. Mitochondrial hsp70 also plays a crucial part in the import process. Mitochondria containing mutant forms of the protein fail to import precursor proteins. The mitochondrial hsp70 is part of a multisubunit protein assembly that is bound to the matrix side of the TIM23 complex and acts as a motor to pull the precursor protein into the matrix space.

Its hard to fathom how natural mechanisms could have 1. discovered the need of import of the proteins, 2. the requirement of a motor, and 3. developed such devide upon the necessity. To do so, forsight, intelligence, planning, a blueprint, and the materials are required to make the motor and the import proteins, and the information where to place it, and how to mount it. The materials would have to be ready available, the individual subparts of the protein and import motor had to be synchronized, the selected parts had to be available all together at the construction site, eventually not simultaneously, but at least when needed, and the parts had to be mutually compatible, that is well matched and able of properly interacting. The subunits had to be put together in the right sequence, in the right order, and interface correctly.
These import proteins and its mechanism is life essential. Without the mechanism functioning right from the beginning of the cell's life, 1. Proteins would be unable to enter into the inner space of the mitochondria, and arrive at their destination where they are required. Thats strong evidence that all had to be previously planned , and put in place fully functioning right from the start.

Like its cytosolic cousin, mitochondrial hsp70 has a high affinity for unfolded polypeptide chains, and it binds tightly to an imported protein chain as soon as the chain emerges from the TIM translocator in the matrix space. The hsp70 then undergoes a conformational change and releases the protein chain in an ATP-dependent step, exerting a ratcheting/pulling force on the protein being imported. This energy-driven cycle of binding and subsequent release provides the final driving force needed to complete protein import after a protein has initially inserted into the TIM23 complex. After the initial interaction with mitochondrial hsp70, many imported matrix proteins are passed on to another chaperone protein, mitochondrial hsp60. hsp60 helps the unfolded polypeptide chain to fold by binding and releasing it through cycles of ATP hydrolysis.

Bacteria and Mitochondria Use Similar Mechanisms to Insert Porins into their Outer Membrane

The outer mitochondrial membrane, like the outer membrane of Gram-negative bacteria , contains abundant pore-forming β-barrel proteins called porins, and it is thus freely permeable to inorganic ions and metabolites (but not to most proteins).
A small section of the double membrane of an E. coli bacterium. The inner membrane is the cell’s plasma membrane. Between the inner and outer membranes is a highly porous, rigid peptidoglycan layer, composed of protein and polysaccharide that constitute the bacterial cell wall. It is attached to lipoprotein molecules in the outer membrane and fills the periplasmic space (only a little of the peptidoglycan layer is shown). This space also contains a variety of soluble protein molecules. The dashed threads (shown in green) at the top represent the polysaccharide chains of the special lipopolysaccharide molecules that form the external monolayer of the outer membrane; for clarity, only a few of these chains are shown. Bacteria with double membranes are called Gramnegative because they do not retain the dark blue dye used in Gram staining. Bacteria with single membranes (but thicker peptidoglycan cell walls), such as staphylococci and streptococci, retain the blue dye and are therefore called Gram-positive; their single membrane is analogous to the inner (plasma) membrane of Gram-negative bacteria.

In contrast to other outer membrane proteins, which are anchored in the membrane through transmembrane α-helical regions, the TOM complex cannot integrate porins into the lipid bilayer. Instead, porins are first transported unfolded into the intermembrane space, where they transiently bind specialized chaperone proteins, which keep the porins from aggregating
They then bind to the SAM complex in the outer membrane, which both inserts them into the outer membrane and helps them fold properly. One of the central subunits of the SAM complex is homologous to a bacterial outer membrane protein that helps insert β-barrel proteins into the bacterial outer membrane from the periplasmic space (the equivalent of the intermembrane space in mitochondria).

Transport Into the Inner Mitochondrial Membrane and Intermembrane Space Occurs Via Several Routes

The same mechanism that transports proteins into the matrix space using the TOM and TIM23 translocators  also mediates the initial translocation of many proteins that are destined for the inner mitochondrial membrane or the intermembrane space. In the most common translocation route, only the N-terminal signal sequence of the transported protein actually enters the matrix space
A hydrophobic amino acid sequence, strategically placed after the N-terminal signal sequence, acts as a stop-transfer sequence, preventing further translocation across the inner membrane. The remainder of the protein then crosses the outer membrane through the TOM complex into the intermembrane space; the signal sequence is cleaved off in the matrix, and the hydrophobic sequence, released from TIM23, remains anchored in the inner membrane. In another transport route to the inner membrane or intermembrane space,
the TIM23 complex initially translocates the entire protein into the matrix space (Figure B).

 A matrix signal peptidase then removes the N-terminal signal sequence, exposing a hydrophobic sequence at the new N-terminus. This signal sequence guides the protein to the OXA complex, which inserts the protein into the inner membrane. As mentioned earlier, the OXA complex is primarily used to insert proteins that are encoded and translated in the mitochondrion into the inner membrane, and only a few imported proteins use this pathway. Translocators that are closely related to the OXA complex are found in the plasma membrane of bacteria and in the thylakoid membrane of chloroplasts, where theyinsert membrane proteins by a similar mechanism. Many proteins that use these pathways to the inner membrane remain anchored there through their hydrophobic signal sequence (see Figure A,B). Others, however, are released into the intermembrane space by a protease that removes the membrane anchor (Figure C).

 Many of these cleaved proteins remain attached to the outer surface of the inner membrane as peripheral subunits of protein complexes that also contain transmembrane proteins. Certain intermembrane-space proteins that contain cysteine motifs are imported by a yet different route. These proteins form a transient covalent disulfide bond to the Mia40 protein (Figure D). 

The imported proteins are then released in an oxidized form containing intrachain disulfide bonds. Mia40 becomes reduced in the process, and is then reoxidized by passing electrons to the electron transport chain in the inner mitochondrial membrane. In this way, the energy stored in the redox potential in the mitochondrial electron transportchain is tapped to drive protein import. Mitochondria are the principal sites of ATP synthesis in the cell, but they also contain many metabolic enzymes, such as those of the citric acid cycle. Thus, in addition to proteins, mitochondria must also transport small metabolites across their membranes. While the outer membrane contains porins, which make the membrane freely permeable to such small molecules, the inner membrane does not. Instead, a family of metabolite-specific transporters transfers a vast number of small molecules across the inner membrane. In yeast cells, these transporters comprise a family of 35 different proteins, the most abundant of which transport ATP, ADP, and phosphate. These are multipass transmembrane proteins, which do not have cleavable signal sequences at their N-termini but instead contain internal signal sequences. They cross the TOM complex in the outer membrane, and intermembrane-space chaperones guide them to the TIM22 complex, which inserts them into the inner membrane by a process that requires the membrane potential, but not mitochondrial hsp70 or ATP (Figure E).

 An energetically favorable partitioning of the hydrophobic transmembrane regions into the innermembrane is likely to drive this process.

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