E.coli: Sec System and Components

 

A majority of the genetic, biochemical, and molecular biological research related to protein translocation and export has been concerned with the Sec translocase components and their functions, individually and cooperatively.

 

There is not one universal pathway setup for all exported proteins to follow.

 

The Sec proteins are dedicated to the translocation of presecretory proteins into the periplasm.

The system of proteins is now known to consist of seven proteins, although only three have been reported as necessary for observed translocationin vitro (Pugsley, 1993).

 

The core of this translocase is a complex between the proteins:

 

SecE and SecY.

 

These integral membrane proteins have numerous membrane-spanning segments and are located in the bacterial inner membrane. They are presumed to constitute a channel and receptor for preprotein passage through the lipid bilayer.

The other protein required for translocation is: SecA.

This is a large protein foundsoluble in the cytoplasm and associated with the inner face of the cytoplasmic membrane. During translocation, this protein binds SecY/SecE, accepts the preprotein, and initiates passage through the membrane.

 

Signal Peptides

 

The overwhelming majority of envelope (periplasmic and outer membrane) proteins found in E. coli are synthesized as larger presecretory forms.

These presecretory proteins haveamino (N) terminal amino acid sequences whose purpose is to direct these proteins for export across the cytoplasmic membrane.

After translocation, these signal sequences are usually removed by enzymes termed signal or leader peptidases.

 

In fact, prokaryote protein export signal peptides have been shown to lead to the translocation of their attached proteins in eukaryotic systems and vice versa (Gilbert, 1990, 1993 ).

 

Unlike in eukaryotes, however, the translocation of these proteins can occur both co-translationally and post-translationally!!!

 

 

Structur of Signal Peptides

It has been shown in E. coli, that a signal sequence alone is unable to target some normally cytoplasmic proteins outside to the periplasm, further indicating that mature protein sequence and structure can also have an effect on translocation!!

 

Signal peptides used in conjunction with the Sec system are exclusively amino-terminal.

 

It has been reasoned that evolutionary pressures rejected C-terminal systems because the mature protein would be synthesized first and allowed to adopt a conformation.

 

They usually contain three key domains:

• A -terminal domain termed N that is usually from 2 to 15 amino acids in size and contains at least one (usually two or three) positively charged amino acid residues. These arginine or lysine residues give the region a hydrophilic net positive charge.
• The next region is termed the H region because it is a fairly long (14-20 amino acids) stretch of neutral, non-polar amino acid residues, often high in alanine and leucine (Oliver). This richness in alanine and leucine leads to the adoption of an alpha-helical conformation. .
• Finally, the C-terminus of the peptide is a six amino acid region beginning with either proline or glycine and predicted to form a reverse turn. The final three amino acids of this C segment are a consensus signal-peptidase processing site, denoted by AXB where B is alanine, glycine, or serine and A is one those three amino acids or valine, leucine, or isoleucine .

 

SecB

 

SecB is a homotetramer that binds specifically to most or a "large subset of" preproteins. The SecB tetramer acts as a molecular chaperone by bindingnewly synthesized preproteins in unfolded, but not native conformations.

The N-terminal signal peptide portion of the presecretory protein is not specifically bound by any regions of SecB and is not required for SecB-preprotein association.

It is suspected that the signal peptide retards the folding of the presecretory protein, allowing SecB to bind multiple sites on the preprotein.

These sites are often beta-sheet domains within the preprotein mature sequence.

This binding of SecB then prohibits preemptive folding of the preprotein into a conformational state that is incompatible with transport through the translocase.

SecB, however, is not influenced by ATP and as such, is functionally different from most chaperone proteins.

SecB does not catalyze folding or unfolding of the newly-produced protein.

Thus, SecB, has the important function of directing the presecretory proteins that it binds to the translocase through a modest affinity with the SecA protein.

 

SecY/SecE/SecG/SecD/

 

SecF

 

SecA is unable to translocate preproteins on its own--it needs a complex of integral membrane Sec proteins.

SecE and SecY are 14 kDa and 48 kDa integral membrane components of the translocase complex that are necessary for translocation. They have 3 and 10 membrane-spanning segments respectively.

The proteins are very homologous and analogous to the yeast and mammal proteins sec61-alpha and sec61-gamma.

Together they likely constitute a hydrophilic pore in the inner membrane that allows for translocation.

SecG is stimulates translocation.

The integral membrane proteinsSecD and SecF have largely been uncharacterized, and only recently have hints to their functions been unearthed.

 

SecY

 

SecY's termini are found on the cytoplasmic side of the bacterial cytoplasmic membrane and the protein's organization is similar to that of the lactose permease coded for by the Lac operon.

Experiments show that the maximum activity is obtained with equalmolar amounts of SecE and SecY. This complex forms a "receptor" for SecA binding.

The likely hydrophilic channel created by the 10 trans-membrane segements.

 

SecG

 

SecG was first identified 1994. It has been known to enhance the translocation activity of the SecY/SecE complex after interation with SecA.

The protein is a 15 kDa integral membrane protein that consists of two strongly hydrophobic membrane-spanning segments and a connecting weakly hydrophobic region between (1996).

The efficiency awarded to the translocase by SecG interaction is hypothesized to be due to alowering of the energy for the insertion and deinsertion cycle of SecA .

 

 

SecD/SecF

 

These two proteins have very similar sequence, which both suggest six membrane spanning segments. These two proteins have been determined to interact with each other.

Both SecD and SecF are needed for effecient protein translocation but it is possible that only a few proteins need SecD and SecF for translocation.

Also, these sec proteins seem important for the maintenance of the electrochemical potential across the cytoplasmic membrane. This evidence suggests that SecF interacts with SecD and SecY in the inner membrane, and that this interaction is important for a late acting step in translocation.

 

Translocation in E. coli via Sec-Independent Secretory Pathways

 

Most proteins in E. coliare translocated by the Sec-dependent mechanism, but a small number of proteins can be translocated across the membrane when the Sec-mediated secretory pathway is experimentally blocked.

 

There are two different types of Sec-independent translocation.

• One involves translocation of various parts of inner membrane proteins by self-insertion. This type of translocation is dictated by the amino acid composition of the translocated segment of the protein and by the composition of the surrounding membrane spanning regions.
• The other type of Sec-independent translocation involves various proteins which have their own dedicated secretory apparatus.

 

Self-insertion

 

Various known proteins can still be translocated across the inner membrane of E. coli when the Sec-mediated pathway is blocked either in strains which carry conditionally lethal mutations in various Sec genes, or by incubation with 2 mM sodium azide.

It seems the Sec-mediated secretion pathway is not efficient on small protein segments.

 

Sec dependence increases gradually with increasing proteins length.

E.g. The phage M13 procoat protein is a short sequence that seems to insert itself into the E coli inner membrane. Elongation of this protein increased its dependence on the Sec machinery as well.

This seems to show that small segments of translocated protein are only Sec-independent because of their length.

Another requirement for self-insertion of shorter segments follows the positive-inside rule. This rule was based on the identification that translocated segment which are not Sec-dependent tend to remain relatively uncharged, while the portion of the protein which is dependent on Sec machinery, or is not translocated at all, is generally more positively charged.

 

Long proteins self-insertion

 

The set of proteins which can self-insert into the membrane which are longer than normal self-insertion proteins segments exhibit other characteristic which are vital to their translocation. This set includes a long 180 residue stretch of MalF and 100 amino acid N-terminus of ProW. These proteins have the special ability to insert themselves into the membrane in spite of their length.

ProW has a 100-residue N-terminus periplasmic segment which exhibits Sec independence. It follows the positive-inside rule, but also has an abnormally large number of negatively charged amino acids on the periplasmic loop.

Inserting positively charged amino acids into the periplasmic segment of the protein inhibits translocation.

Proton-motive force aids in the translocation of negatively charged amino acids, and hinders positively charged amino acids. This could be one way the extra long loop of ProW translocates itself across the membrane.

A mechanism of self-insertion of Sec-independent proteins is suggested which is dependent on aid from neighbouring hydrophobic, transmembrane regions.

 

Translocation in E. coli via Sec-Independent Secretory Pathways

 

A small number of proteins are known to have their own export mechanisms.

 

These export apparatuses, involved in secretion of each protein, are a member of a larger set of transport proteins called the ATP-binding cassette (ABC) proteins. Within this family of ABC proteins is a smaller subset of specific transport proteins who are members of the 'MDR-like' subfamily of proteins. The best known member of this group is P-glycoprotein or MDR protein, which is responsible for multi-drug resistance in tumor cells. The specific export for hemolysin is characteristic of the general secretory pathway for the following "MDR-like" export systems.

Colicin V

Colicin V has a unusual amino acid composition, with 17% gly, 14% Ala and 10% Ser, which seems to make it a very flexible protein. Its export across the plasma membrane involves an "MDR-like" mechanism.Colicin V does not posses a normal N-terminal signal sequence. It has a large hydrophobic region in the N-terminal half of the proteins, which could be involved in its insertion into the membrane of the target cells, where it acts to kill the cell. It also possesses an uncharacteristic net negative charge at the N-terminal end. The first 39 residues of colicin V are important for successful translocation. CvaA and cvaB seem to be involved with its export and or secretion. It was found that cvaB is analogous to other proteins in the "MDR-like" subfamily involved in the export process. CvaB has six hydrophobic regions whereas cvaA seems to be analogous to HlyD in hemolysin as a second component of the export process.

Hemolysin

Hemol ysi n (Hl yA), a 102 4-re sid ue pro tein, doe s not con tain a nor mal N-ter min al signal sequence, but has been shown to have a C-terminal signal sequence instead. It seems that the last 48 amino acids of the HlyA protein contain a functional signal sequence domain. Deletion of this region showed that the protein maintained full expression, and stability.

3 characteristic features:

• A potential amphipathic alpha-helix
• From Aa 997-1024, an 13 amino acid uncharged sequence and a beta-sheet.
• A 52-amino acid C-terminal sequence was able to direct wild-type secretion, indicating that the sequence is important as a signal peptide.

Secretion of HlyA out of the cell is dependent on two closely related genes hlyB and hlyD. This pathway also seems to involve TolC for secretion out of the outer membrane.

HlyB might by an adhesion domain for the inner and outer membrane.

The most obvious feature of this complex is the ATP-binding domain. This domain has very high sequence identity with the "MDR-like" export proteins. These molecules function as pumps.

The HlyD protein may give the export mechanism specificity for translocation of hemolysin only.