Protein targeting

 

• Proteins possessing a signal sequence trigger extrusion of these proteins through the endoplasmic reticulum membrane (ER).
• Membrane proteins become embedded in the phospholipid bilayer, while water soluble proteins may either be secreted or remain in the lumen of a particular organelle.
• Sorting sequences & signals subsequently target the proteins to different cellular components (e.g. lysosomes).
• Retention sequences & signals ensure that permanent residents of a particular compartment are retained there.
• It is thought that proteins which do not contain a sorting and retention signal are by default secreted from the cell, or if they posses a membrane anchor, get targeted to the plasma membrane.

During their synthesis - which commences in the cytoplasm - membrane proteins, lumenal proteins and secreted proteins (but not cytoplasmic resident soluble proteins e.g. haemoglobin) are found to posses a signal sequence of 15-30 amino acids at the N-terminus.

After synthesis of these proteins is completed the signal sequence is normally cleaved off by a specific protease (signal peptidase).

Signal sequences

 

The signal sequence has an affinity for an RNA/protein particle known as the signal recognition particle (SRP).

Signal sequences show little sequence similarity, but rather a structural similarity. They contain a:

• short basic amino terminus followed by
• a segment of 10 (3) hydrophobic amino acids.

 

 

Examples of signal sequences

 

MATGSRTPWLQEGSA*FPT Human Growth hormone

MALWMRGPDPAAA*FVN Human Pro insulin

MAAKSASAATA*SIF Zea Maize Protein

MKAKG*DQI Human influenza virus

Cleavage at the site marked with the asterisk is carried out by a signal peptidase located on the lumen side of the endoplasmic reticulum.

 

Signal recognition particle

 

Translation of membrane, luminal and secretory proteins begins on free ribosomes in the cytoplasm.

When 70-80 amino acids have been polymerised and the signal sequence emerges from the ribosome, it is recognised and bound by the 54kDa protein of the Signal Recognition Particle (SRP).

 

The SRP in eukaryotes consists of six different proteins (9K, 14K, 19K, 54K, 68K and 72K) tightly bound to one molecule of RNA (7S RNA) about 300 nucleotides long.

 

PROTEIN TRANSLOCATION

 

• When the signal peptide binds to the SRP it delays or even arrests translation until the complex encounters an integral membrane protein complex found only in the ER: the SRP receptor, sometimes referred to as the ``docking protein''.
• After ``docking'' of the complex on the ER, the SRP detaches from the complex and returns to the cytoplasm.
• The unfinished (``nascent'') protein still attached to the ribosome now binds to other proteins in the ER.
  The nascent chain is inserted into a protein translocation tunnel in the membrane and the ribosome becomes membrane bound through its association with ribosome receptor protein(s) in the membrane.

The SRP has two functions: ittargets nascent proteins to the endoplasmic reduction and it keeps the signal sequence segregated from the rest of the polypeptide chain and prevents premature folding.

 

• Protein synthesis resumes with the ribosome now attached to the ER and the protein is ``threaded'' through the translocation tunnel.
• Translocation across the ER membrane is initiated while the protein is still being synthesized, i.e. co-translational translocation.

Stop-transfer sequences

 

• Hydrophobic stop-transfer sequences in the sequence of integral membrane proteins halt the translocation process and thereby ``fix'' and orient these proteins in the bilayer.
• Secreted and lumenal proteins lack such stop-transfer sequences and are therefore completely extruded into the ER lumen.

The transmembrane topology of single-span (bitopic) and multi-span (ploytopic) membrane proteins can be explained by the combined function of signal peptidase, signal sequences, and internal signal sequences.

 

• Thus, the only difference between secreted & lumenal proteins versus membrane proteins is the presence of a stop transfer sequence.
• In fact many proteins are known to be alternatively spliced whereby one splicing isoform is membrane bound and the other is secreted from the cell. (Antibodies are famous examples of such an occurrence.)

 

Intracellular targeting

 

For lumenal, secreted and membrane proteins the ER represents the point of origin in their cellular ``travels'' towards their final destination.

 

• Note that for many proteins the ER may represent the final destination.

A point of view held by most scientists until very recently is that a bulk flow of ``material'' exists from the ER towards the plasma membrane.

Therefore, any proteins residing permanently in other cellular locations are either retained at one point during the flow by specific interactions, or switch to another route ``off the beaten path'' once again as a result of a particular signal.

Although there are now many who hold the view that no bulk or default flow exists.

 

Transport to the plasma membrane

 

After undergoing all of the modifications, proteins leave the ER in transport vesicles which bud from specialised ribosome-free regions situated adjacent to the Golgi apparatus and called transitional elements.

 

From here they are delivered to the cis face of the Golgi after which a host of Golgi specific modification ensues, most notably glycosylation modification (see above).

The proteins are then ``passed'' through the medial Golgi to the trans-Golgi-network (TGN). Once again two views are held as to how this process is achieved:

• Repeated cycles of vesicle budding and fusion from one Golgi cisternae to another: cis medial trans-Golgi.
• Maturation of the Golgi cisternae themselves, i.e. the cis Golgi becomes the medial Golgi and likewise the medial Golgi becomes the trans-Golgi.

Subsequently proteins leave the trans-Golgi network to the plasma membrane either:

• In vesicles which bud from the trans-Golgi network (if you subscribe to vesicular traffic between the Golgi Cisternae)
• Or through the entire trans-Golgi network disintegrating into small vesicles that are then transported to and fuse with the plasma membrane.

 

Transport to the places other

than the plasma membrane

 

Whether or not we believe that there exists a bulk flow of material from the ER towards the plasma membrane, we do know of a variety of targeting signals that cause proteins to either be retained in a particular compartment or to be selectively recruited there.

The difference between retention and recruitment is not subtle but rather reflects dramatically different mechanisms.

• Selective retention along a particular pathway states that the proteins do not leave that compartment due to a particular interaction with residents elements. This signal will then be regarded as a retention signal.
• Active recruitment of proteins to a particular compartment suggests that the proteins continue along the ``bulk'' flow of things, but then get retrieved along a retro-grade pathway. This signal will then be regarded as a retrieval signal
• Sorting signals are signals that ``divert'' a protein along the ``bulk'' flow towards an alternate path such as targeting of the proteins to the lysosome.
• An additional mechanism might exist for specific localization which does not involve targeting at all, but rather selective degradation of a particular protein in all places but its destined origin. Example for this mechanism are few but do exist.

 

Examples of retrieval, retention & sorting signals

 

• The signals that designate particular proteins as ER proteins are mainly retrieval signals. They operate by way of a``recycling'' pathway from the cis-Golgi, in which proteins containing the particular signal are selectively inserted into vesicles budding from the cis-Golgi, that later fuse with the ER.
• K(X)KXX signal: double lysine residues occurring within the last 5 amino-acids of the proteins in type I membrane proteins. Therefore the signal is in the cytoplasm.
• RR signal: double argenine residues occurring within the first five amino-acids of a type II membrane protein. Therefore the signal is in the cytoplasm.
• KDEL signal: a tetra peptide in the carboxylterminus of the protein. This signal should be found in the lumen of the ER

 

• Signals for Golgi localisation are of the retention ``variety'', and their mechanism of action is less understood then ER localisation signals.
• Retention through oligomerisation is thought to take place in the Golgi. Large aggregates or hetero-oligomers are thought to be selectively retained in the Golgi. The interactions are thought to take place between the membrane spanning domains of the oligomerizing proteins.
• Retention through membrane thickness. The length of the transmembrane -helical segments of many resident Golgi proteins, has been shown to be significantly shorter than their plasma membrane counterparts. It was also shown that truncating the transmembrane segments of many plasma membrane proteins would result in retention in the Golgi.

 

• Targeting of proteins to thelysosome is achieved by a sorting signal of phosphorylation of manose sugars in the oligosaccharide chain: manose 6-phosphate.

NOTE: The relative ``strengths'' of the signals listed above can be measured when contradictory signal are inserted in the same proteins.

 

Molecular targeting to endosymbiontic vesicles

 

Fewer than a dozen mitochondrial proteins are encoded by mitochondrial DNA and synthesized inside mitochondria. Almost all of the latter are not complete enzymes but are subunits of multimeric enzymes.

Many cytoplasmically-coded mitochondrial (& chloroplast) proteins (membrane-bound and soluble)are completely synthesised on free cytoplasmic ribosomes as larger precursors.

The extra segment which is analogous to the signal peptide, binds to organelle specific receptors in the target membrane (e.g. mitochondrial outer membrane).

 

• Thus all species within a class to be translocated (to mitochondria or chloroplasts) are synthesized with class-specific functionally identical signal sequences!!
• Binding of receptor to signal sequence results in creation or opening of a pore that allows passage of one polypeptide at a time.
• The targeting sequences of proteins imported into mitochondria (except outer membrane proteins), are 20-80 residues long and at the N-terminus.
• They are removed by a signal peptidase in the matrix after import.

 

Targeting sequence:

can form amphipathic helices which readily insert into membranes. (Notice the regularly repeating positive charged residues in this cytochrome oxidase targeting sequence.)

 

Expenditure of energy (both ATP and the membrane potential) is needed for import of mitochondrial proteins except those resident in the outer membrane.

 

Mechanism: Import into mitochondria

 

• The evidence indicates that the targeting sequences bind to receptors in the mitochondrial outer membrane.
• As a result of lateral movement of these receptors, complex forms between the receptors and ageneral insertion protein (GIP) which in effect "collects" precursor proteins and inserts them into the outer membrane.
• ATP is required for this insertion step, probably to unfold the protein into a translocation competent form.
• The translocation route is localized at points where the inner and outer mitochondrial membranes are attached ``contact or adhesion sites''.
• The precursor proteins are thought to be translocated through a proteinaceous tunnel in an unfolded state as in the ER.
• The membrane potential is required for the initial entrance of the targeting sequence into the matrix through the inner membrane.
• Once inside the mitochondrial matrix, ATP-driven ``unfoldases'' and ``refoldases'' are involved in the reversible formation of the `translocation competent'' state of the protein, as was the case in the ER lumen.
• The mitochondrial heat shock protein (HSP70) binds to the precursor proteins emerging on the matrix side and thereby supports the completion of translocation.
• The targeting sequences are cleaved off by the enzyme mitochondrial processing peptidase (MPP) in co-operation with the processing enhancing protein (pep).
• Imported proteins destined for the matrix are refolded in an ATP dependent association with the heat shock protein HSP60.

 

Import of proteins from the cytosol to the inner-membrane or to the inter-membrane space of mitochondria

 

• Import of proteins from the cytosol to the innermembrane or to the inter-membrane space requiresmore than one targeting sequence.
• These proteins destined for the inner membrane or the inter-membrane space will have hadnew inner membrane targeting sequences for these destinations exposed by MPP cleavage of the initial matrix targeting sequence.
• These newly exposed targeting sequences are then recognized by an inner membrane translocation apparatus (proteinaceous tunnel?) and re-translocated (after ATP dependent interaction with HSP60).
• Transport into the inter-membrane space requires a third step in which a protease with its active site in the inter-membrane space, cleaves the bulk of the protein from the anchored signal peptide.
• When mitochondria are ``poisoned'' with inhibitors (cyanide etc.) precursor forms of mitochondrial proteins still bind to receptors on the outside of the outer mitochondrial membrane, but are not translocated. If the inhibitor is removed, translocation resumes.

Proof that the cleaved presequence of mitochondrial proteins comprises an organelle-specific signal for uptake comes from experiments in which non-mitochondrial presequences were translocated into the mitochondria when authentic mitochondrial presequences were fused to them.