Protein Trafficking


Protein trafficking begins with targeting of proteins to their proper destinations. This is carried out on the Cis-side of the membrane (cytosolic) and is handled during (co-translational and SRP-dependant) or shortly after synthesis (post-translational and SecB-dependant) of the protein on the ribosome.

Targeting of a protein to its final destination is controlled by its signal sequence or lack thereof (retention in cytosol).

ER signal peptide

mitochondria presequence

chloroplasts transit peptide

Each signal sequence is quite different from the others and quite incompatible to ensure consistent targeting to the correct compartment.


The Signal Recognition Particle (SRP)

The SRP is a complex of RNA and protein components. It binds to the N-terminal signal sequence of a nascent protein chain just as it exits the ribosome and halts further translation. When the ribosome-SRP complex binds to a SRP receptor, the ribosome and nascent chain are brought to a translocon.

The 6 protein subunits of the eukaryotic SRP are organized into two functional domains: a smaller one and a larger one. The smaller domain is responsible for translational regulation (halting), while the larger domain is responsible for signal sequence recognition and binding to the SRP receptor. SRP54, found in both the eukaryotic and prokaryotic SRP is the critical subunit that recognizes the signal peptide of the nascent protein chain.

The SRP cycle of the diagram and more SRP information)

When ribosomes in the process of translation have translated the N-terminal signal sequence of a nascent protein chain, signal recognition particles (SRP) associate with those ribosomes. Translation is halted until the resulting ribosome-SRP complex interacts with the SRP receptor on the target membrane. The ribosome then interacts with a translocon (Sec61 in the case of the eukaryotic ER), inserting the signal sequence into it and continuing translation to translocate the protein across the membrane. The SRP dissociates from the ribosome and the receptor and diffuses back into the cytsol to begin the cycle anew.

Ribosomes are either "free" or "bound" and make proteins destined for various parts of the cell.  The ribosomes themselves are most likely identical in structure and switch from the "free" to the "bound" conformation alternatively.  The secretory proteins are maked with a specific "signal sequence"  of approximately 20 amino acids.  The sequence allows the ribosome to attach to a receptor on the surface of the rough ER.  The signal sequence is removed by an enzyme as the maturing polypeptide chain grows into the ER lumen.  If the mRNA lacks a segment coding for the synthesis of the ER signal, the ribosome will remain "free" in the cytosol.

Rough endoplasmic reticulum has ribosomes for protein systhesis.  Newly synthesized proteins are stored in cisternae.  Proteins are then sent to the Golgi by small vesicles, or inserted into the membrane if the proteins are membrane proteins.  Rough ER can be either vesicular or tubular.  The ribosomes lie on the outer surface of the cisternae.

Protein translocation can occur in a cotranslational manner (ribosome tightly bound at the membrane) or can occur post-translationally. In the cotranslational mode, the ribosome-bound nascent chain is targeted to the translocation sites in the ER membrane by the assistance of the SRP (signal recognition particle) RP) and its membrane receptor in a GTP dependent manner.  The 54 kDa subunit of SRP recognizes the signal sequence.  The "nascent chain associated complex" is thought to be a factor in the correct targeting of the nascent chain complexes to the membrane.  Very little is known about the mechanism - Cytosolic HSP70's are thought to be involved, and it is not known if a similar signal sequence exists for recognition like the SRP-dependent targeting system.


The sec61 integral membrane protein is composed of alpha, beta, and gamma subuits (SecA, B, E, G, and J in prokaryots.)  This forms the central translocation complex through which the the protein is inserted.  When the transmembrane segment first enters the translocation channel it closely associates with sec6alpha, and as elongation continues, it moves into the hydrophobic regions between the Sec61alpha, beta, and gamma trimers.  At this point, the protein can be released into the bilayer.  Proteins can also be transported from the membrane back through the translocation channel to the cytoplasm.

SecB is a molecular chaperone found in bacteria that mediates translocation of nascent chain polypeptides across the cytosolic membrane.  Crystal structure of SecB indicates the molecule is a tetramer that is a dimer of dimers.  Two long channels transverse the molecule.  They are bound by flexible loops with hydrophobic amino acids.  There is an acidic region at the top of the molecule that is thought to play a role in binding to SecA.  SecB prevents folding inside the cell for precursors of exported proteins and delivers them to SecA (ATPase of the translocation apparatus).

This illustrates again how SecB functions as a chaperone and delivers the protein to SecA and the translocation apparatus.

The translocon is the complex of proteins that are associated with the translocation of nascent polypeptides into the lumen or cisternal space of the endoplasmic reticulum.  It functions in regulating ribosomal/ER interaction, translocation, and integration of membrane proteins in their proper orientation.  Signal peptidase, TRAM, and signal recognition protein are asscociated with the translocon.

The translocon consists of three or four sec61 complexes, each composed of three proteins (sec61 alpha, beta, and gamma).   The alpha protein is a transmembrane protein (ten membrane crossings).  The TRAM protein crosses the membrane eight times.

Model for GTP utilization by SRP54. In this model, SRP54 is proposed to undergo a series of sequential confromational changes that drive SRP unidirectionally through cycles of protein targeting.  T = SRP54-bound GTP;  D = SRP54-bound GDP.


    Domain structure for the three GTPases in SRP and SRP receptor.  (A)  The ras-like GTPase domain is characterized by four conserved sequence motifs (G-1 to G-4).  Note that the GTPase domains of SRP54 and SRa are related to one another (shaded box) and that this homology extends through the N domain.  The transmembrane region of SR-beta is indicated (TM).  (B) The M domain of SRP54 is linked to the N/G domains by a protease-sensitive hinge region.  The N and the M domains are likely to be in close spatial proximity because the N and C termini of ras are in close proximity in the folded protein. (C)  The heterodimeric SRP receptor is likely to be anchored in the membrane by the single transmembrane segment of SR-beta.  The N/G domains of SRa are linked to SR-beta by a protease-sensitive hinge region.  It is not known how the N-terminal region of SRa contacts SR-beta, nor whether it also contacts the hydrophobic core of the membrane.

Three possible arrangements of the signal sequence  and nascent chain at an early stage of translocation across the ER membrane.  A 64-residue preprolactin nascent chain is shown schematically.  (A) in an aqueous pore (white) through the apolar core of the bilayer (light gray); (B) in an aqueous pore (white) through the apolar core of the bilayer (light gray); and (C) embedded in the hydrophobic interior of the membrane.  The dark grey shapes represent ER-membrane proteins that have been shown by photocrosslinking to be adjecent to the nascent chain during translocation.  The small black ovals on the nascent chain represent the approximate locations of the two fluorescently labeled lysines in the preprolactin signal sequence, four and nine residues from its amino-terminal end.

Following SRP-dependent targeting to the ER membrane, the signal sequence and nascent chain are initially located in an aqueous space that is sealed off from both the cytoplasm and the lumen of the ER.  After translocation at the membrane has increased the length of the nascent chain to about 70 residues, the aqueous pore through the ER membrane is opened to the lumen.  Only one of the four different mechanisms for opening or creating a pore is shown here.  In this figure the pore is presumed to be closed by a lumenal protein such as BiP that binds to and blocks the end of the pore early in translocation.  The pore is then opened when the nascent chain becomes sufficiently long to interact with the lumenal protein and trigger its release.

Top view of the aqueous translocon pore in an intact translocation intermediate.  The charcoal ring represents the ER membrane proteins of the translocon that surround the aqueous pore, while the grey region represents the ER membrane phospholipids in the plane of the figure.  The black circle represents a fully extended polypeptide that has a hydrated diameter of ~11A and is oriended perpendiculary to the plane of the membrane.
(A) The pore is shown with one strand of the hydrated nascent chain and one hydraged NAD+ (black rectangle).
(B) The pore is shown with an extended nascent chain and the antigen binding site (stiped oval) at the end of an elongated Fab molecule (black oval) inserted into the translocon.

The translocon pore is precisely gated during Type I membrane protein integration.
Following SRP-dependent targeting to the ER membrane, the nascent chain is initially located in an aqueous channel that is sealed off from both the cytosol  by the tight ribosome-membrane junction and also by the lumen by the as-yet-unidentified gate.  After the nascent chain length increases to about 70 residues (I), the aqueous translocon pore is opened to the lumen to allow the exocytoplasmic domain to enter the ER lumen.  The TM segment of the nascent chain is completely synthesized without chainging translocon pore accessability (ii), but after the C-terminal end of the TM segment is 4 residues from the tRNA, the lumenal end of the pore is completely closed (iii).  After five more residues have been added to the nascent chain, the ribosome-membrane junction is completely open (iv).  Although the figure depicts a very large movement of the ribosome, the actual conformational change may be much smaller.  The signal sequence is also shown in the pore after step (iv), but it may have already left the pore.  Steps (iii) and (iv) occur while the TM segment is still inside the ribosome.  The TM segment reaches the translocon in stem (v), and the signal sequence has been removed, thereby leaving the N-terminal end of nascent chain in the ER lumen.  The nascent chain of this signal-spanning membrane  protein then remains exposed to the cytoplasm as the translation continues (vi) and until translocation terminates and integration is complete (vii).

Model of post-translational secretory protein translocation into the yeast ER: component mediating polypeptide targeting, insertion, and maturation.

The protein translocation systems of the ER, the mitochondrial inner membrane, and the bacterial plasma membrane.  Export systems are in green, import systems in red-brown.  NTP-coupled protein translocation motors in red, cytosolic spaces in light blue or gray, and extracytosolic spaces in tan.  The two systems of the ER represent the posttranslational system identified in yeast and the cotranslational system.  EF, ribosome-associated elongation factor of protein synthesis; Sec63c and Sec61c stand for the respective hetero-oligomeric complexes; Tim, transport components of the mitochondrial inner membrane.  BiP (Hsp70) may also be required for cotranslational translocation into the ER.  The SecA domain surrounded by a dotted line is involved in the insertion-deinsertion cycle during the motor function of this protein. N indicates an N-terminus generated by proteolytic removal of the presequence.

Three possible ways to drive protein translocation across membranes.
(A) Translocation elongation drives the nascent chain through the membrane pore; (B) an ATP-consuming enzyme moves the polypeptide chain through the membrane pore; and (C) ATP-driven cycles of chaperonin binding and release pull the polypeptide chain through the membranepore.  It is possible that two, or maybe even three of these mechanisms collaborate to translocate or integrate certain proteins.

Vesicle transport

Proteins are taken from one compartment to another via gated transport (red), transmembrane transport (blue), or vesicular transport (green). The amino acid sequence that defines the protein is what actually directs its transport. Along the way, decisions are made as to where the protein must eventually travel; these decisions are made at the boxes in the figure.

The movement and direction of proteins is an integral and dynamic portion of cellular biology.  Without the ability to move and direct the proteins as they are synthesized, the cell would be in an utter state of confusion, not to mention the scientists that must catalogue and describe the processes.  This form of order is seen throught all species in some form or another.

The DNA contained in the nucleus is transcribed into mRNA which leaves the nucleus only to meet the rough endoplasmic reticulum. Here the mRNA is translated into a protein, which is packed and shipped to the golgi apparatus and eventually to its destination in the cell.  As one can see, the sites of packing and modification are photographed to high resolution.  Note the ribosomes on the rough ER and the lack thereof on the smooth ER.  The golgi complex is modular and shaped such that there is a direction of progression for maturing proteins.

Budding and fusion of the lipid bilayer is responsible for the movement of concentrated amounts of certain proteins and molecules. It is this process that allows for virtually all vesicular transport in the cell.  The process of budding and cell adhesion and the actual incorporation of a vesicle into the cell membrane of an adjacent organelle is a detailed and complex process.

This picture shows the best-understood pathways of protein sorting in the trans Golgi network.


Selected and nonselected vesicular transport in nonpolarized cells.
Nonselected (constitutive) transport (blue arrows) is postulated to be mediated by coatomer-coated vesicles, while various forms of selected (signal-mediated) transport (red arrows) are postulated to be carried out by clathrin-coated vesicles.  In polarized cells an additional signaled pathway from the trans Golgi network is required


A-D are models of biomembrane fusions. The process is carried out by certain molecules called fusion peptides. These proteins pull two pieces of membrane into a hairpin structure, one that is inherently unstable. As the two membranes get closer together, the hydration sphere is lessened, thus allowing the unfavorable bends to become flattened as the cell membranes merge, creating one cell.  The diagram is an example of viral pore formation.  When HA, the rod-like structure, stays vertical, the fusion peptides are extruded sideways and induce a semi-fusion intermediate seen in A.  This evolves into a lipid-lined fusion pore seen in B.  When HA tilts, the fusion peptides penetrate the adjacent cells and create a stalk-like intermediate that collapses, after the removal of the hydration sphere, into a pore.  Without these fusion peptides, the docking of these two membranes would not be possible.

This cartoon depicts the many membrane fusion events that take place in cells. Fusion events are common in cells, and are necessary for the survival of the cell. Problems with fusion can result in a cells incapacity to do its job in the organism.  Imagine for a moment that the ability for membranes to fuse was postponed for five minutes in your body.  Would you still be alive?  Probably not.  Imagine if that ability were halted for five seconds.  Would you still be alive then?  Probably not.  Now imagine that your body's ability to fuse membranes was halted for five microseconds.  Would you still be alive.  Perhaps.  The capacity for an organism to fuse membranes is paramount for their survival.

Without the ability for cells to undergo fusion and fission, the capacity of the organism to undergo cell division and growth would be limited. The cell cycle picture above depicts very well the events needed for an organism to grow. Included in this is the ability for cells to fuse and break apart, or undergo cytokinesis.

Another important feature of protein trafficking is the ability to recycle lost peptides, rather than resynthesize what is lost. Valuable energy is saved by having a salvage system. So called salvage receptors allow the return of misdirected peptides and proteins.

Targeting of peptide and protein containing vesicles is necessary for proper delivery. v-SNAREs are often incorporated into the membrane of the vesicle, intended to meet and dock with t-SNAREs of the intended target organelle. Since v- and t-SNAREs bind with such strong affinity, the vesicles are recognized and incorporated into the target organelle with speed and accuracy.

The donor organelle creates a transport vesicle that is coated with polymers that indicate that the vesicle is bound for transport.  Shortly, the coat is removed and the vesicle approaches the target organelle.  Tethering is needed for proper positioning, but docking is where the true fusion begins.  v-SNARE and t-SNARE will anneal with very high specificity, thus allowing the docking and fusion of the vesicle with its target organelle.  Below is a picture describing docking and fusion in gross detail.

As the vesicle approaches the target organelle's lipid bilayer, the tethers interact with the target organelle's cytoplasmic proteins.  This ensures that there will be proper docking.  As the vesicle moves closer, it is allowed to come into close contact with the target organelle.  At this point the two membranes are touching their polar head groups.  Water is tightly associated with the polar head groups.  As this water is squeezed out of the way, or as the hydration sphere is lessened, the lipid-lipid interactions take over and the hydrophobic domains begin to merge between the vesicle and the target organelle's lipid bilayers.

Snares work by coordinating the forces of cohesion, adhesion, and an actual physical pulling mechanism in order to fuse the vesicle with the membrane.  In this example, VAMP and SNAP-25 are working as the above models imply with V-SNARE and T-SNARE.  Very similar interactions allow the vesicle membrane and the plasma membrane to delete the hydration sphere and fuse the membranes.

A good example of vesicle fusion with the plasma membrane is depicted by the following:
The image is avaliable at


The v-SNAREs and the t-SNAREs interact in such a way that the vesicle is forced down and held tightly against the target organelle's lipid bilayer.  And as the hydration sphere is lessened, a hemifusion intermediate is formed.  This is an unstable structure that will fall apart if not converted to full fusion or reverted through fission.  Since the SNAREs are tightly associated, the hemifusion intermediate gives way to true fusion.  The vesicle now becomes part of the target organelle.  Since the lipid bilayer is fluid, local proteins can freely diffuse across and occupy the area that once was the vesicle.

GNRP (guanine-nucleotide-releasing protein) is responsible for the insertion of ARF-GTP. The ARF-GTP is then able, once inserted to the outside of the vesicle, to direct the addition of the coatomer, creating a coatomer-coated vesicle. This ensures secure packaging of peptides and proteins into vesicles and proper delivery. Often the coat falls off the vesicle after it leaves the vicinity of the originating organelle.

The coat is recycled shortly after budding and movement of the coated vesicle toward the target organelle. Inside the vesicle, holding the cargo, is the receptor. It too is recycled in a fashion similar to the delivery.  As in the case of the golgi apparatus, the coat proteins that allow vesicles to move from left to right are CopII and the proteins that allow vesicle recycling, or movement of vesicles from left to right are CopI.

This depicts another view of proteins leaving the nuclear envelope to enter the rough ER. They then move to the cis-Golgi and to the trans-Golgi. Later they are transported via secretory vesicle to the target cell or organelle. Also, as in the earlier picture, if there are problems, the peptides are placed in a vesicle and are bound for the late endosome and eventually to the lysosome to be degraded.

For more information on the subjects presented above, it would be wise to visit the following websites:




Enough shameless promotion, here's some sites that are of paramount interest:

1. - A site dedicated to the scientist studying cells

2. - A site concerned with SNAP and SNARE mediated vesicle fusion

3. - An excellent site for biochemistry related words and references

4. - A page from the University of York, used to supplement the information in this page

5. - For more information on signal sequence on secretory proteins