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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
Mitochondrial import
signals.
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.
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.
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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 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.
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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.
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