Mitochondrial replication is diagrammed in the cartoon. Mitochondria replicate much like bacterial cells. When they get too large, they undergo fission. This involves a furrowing of the inner and then the outer membrane as if someone was pinching the mitochondrion. Then the two daughter mitochondria split. Of course, the mitochondria must first replicate their DNA. This will be discussed in more detail in the next section. An electron micrograph depicting the furrowing process is shown in these figures. The figure on the right was taken from Fawcett, A Textbook of Histology, Chapman and Hall, 12th edition, 1994.   Sometimes new mitochondria are synthesized in centers that are rich in proteins and polyribosomes needed for their synthesis.

Mitochondria have their own DNA and Ribosomes
Mitochondria have some of their own DNA, ribosomes, and can make many of their own proteins. The DNA is circular and lies in the matrix.in punctate structures called "nucleoids".  Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA). Is it normal to have areas with many mitochondria?  Yes in some cells and situations.  An example would be the hummingbird flight muscle which has many mitochondria and many cristae in the mitochondria. What would happen if a mitochondrion could not undergo fission?  It would continue to grow and grow and eventually become a huge organelle.  Mutant yeast can be produced in which mitochondria are unable to divide.

When is replication not normal?  Below shows replication and production of cristae in response to defective mitochondria and low ATP.  If ATP is low, the cell is stimulated to make more mitochondria.  But, if they are defective as in the lower photograph, ATP cannot be produced.  To compensate, mitochondrial will make more cristae (see the mitochondrion on the right.

Mitochondrial DNA and Ribosomes.

The ribosomes can actually be visualized in some mitochondria.They are seen in the matrix as small dark bodies. DNA can also be visualized in mitochondria. The DNA is circular and resembles that of a bacterium in its basic structure. The micrographs show the DNA and ribosomes in a close-up view. Note that the circular structure of the DNA is not evident. It is noted by an arrow. There are two sets of ribosomes seen, each is circled. One can use specific inhibitors of each site to differentiate the sources of these proteins. This is shown in the following figure. Note that antibiotics inhibit mitochondrial protein synthesis (similar to that of bacteria). The nuclear coded proteins must then be imported into mitochondria, which is the subject of the next section.

Mitochondrial import signals.
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.  There must also be positive charged amino acids near the entry point.  

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".  That means it must be uncoiled.  This requires energy, as shown in the following cartoon (Steps 1 and 2). The mitochondrial proteins use 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. 

Step 1: Protein unfolds as it binds, this  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.

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.  

The entry point 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.  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 .  

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" , 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.

Characteristics of Tim Complexes
Proteins 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-hsp70 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 . 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. 

Entry of proteins is dependent on the hydrogen pumps (electron transport chain)
 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 GIP. The protein then enters the matrix where the cleavable preprotein is clipped off by a protease and a chaperone protein ( mt-hsp70) in the matrix works with Tim44 to complete the full transfer to the matrix. This chaperone 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. Negative charges in the matrix, set up by the pumping of hydrogen ions to the inter cristal space, attract the protein which has positive charges on the end that enters the GIP. Some mitochondrial proteins destined for the inner membrane have a cleavable signal peptide followed by one or more  membrane-spanning segments that  serve to insert the polypeptide into the inner membrane after it gets in the matrix. This is how the electron transport proteins get into the inner membrane. Return to Menu

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".. 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. 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.  The mitochondria were characterized by few cristae.

Mitochondrial Inheritance In mammals, 99.99% of mitochondrial DNA (mtDNA) is inherited from the mother.  This is because the sperm carries its mitochondria around a portion of its tail and has only about 100 mitochondria compared to 100,000 in the oocyte. As the cells develop, more and more of the mtDNA from males is diluted out.  Hence less than one part in 10⁴ or 0.01% of the mtDNA is paternal.  This means that mutations of mtDNA can be passed from mother to child.  It also has implications if one does cloning of mammals with the use of  somatic cells.  The nuclear DNA would be from the donor cell, but the mtDNA would be from the host cell.  This is how Dolly the sheep was cloned.  The following diagram shows a family in which the mother passed the mutation to her three children, but only the daughters passed it to subsequent generations.

Mutations in mammalian mtDNA do cause diseases, because there is such a short sequence and very heavy information content in the sequence.  Since each cell contains hundreds of mitochondria and thousands of copies of the genome, the effects of the mutated mitochondria may be diluted out.  As expected, those tissues or organs most likely to be affected would be the ones most dependent on oxidative phosphorylation (ATP production). In young persons it might not be picked up because even a person with 15% normal mitochondria might have enough to be healthy.  However, aging patients may show a more severe disease phenotype.    Also, the mother passes the mutation unevenly to the different daughter egg cells.   This uneven distribution may result in some children with lethal forms of the disease and others with mild forms.

What happens to old, worn-out mitochondria?
Mitochondrial numbers are controlled by autophagy. This is a process by which lysosomes are involved in controlling cell constituents. The process begins by wrapping endoplasmic reticulum membranes around the mitochondrion. Then, vesicles come from the Golgi complex and join with the autophagic vacuole. These vesicles contain hydrolases attached to the mannose 6 phosphate receptors in the vesicle membranes. The lysosome web page discusses their function and fate. Recall that they fuse with the autophagic vacuole.   The acid pH then allows the hydrolases to be removed from their receptors. The receptors are recycled back to the Golgi complex in other vesicles.
Learn more about mitochondrial structure and function
Learn about export from mitochondria    Return to Menu