Protein Processing I: Getting things where they need to go -Targeting of Proteins and Vesicles

The key concept:

Proteins are synthesized in the cytosol compartment and are targeted to many sites in the cell: mitochondria, chloroplasts, endoplasmic reticulum, nuclei etc. Vesicles that bud off of the plasma membrane, the endoplasmic reticulum or the Golgi apparatus are also targeted to specific sites in the cell. How does this happen?. The basic idea is very similar to that underlying the postal service:

Figure 14-5

Each protein contains  information in amino acid sequences that serve as an address. All proteins targeted to the same destination, e.g. nucleus, carry the same address signal encoded in their protein sequence.  There is a specific protein receptor that corresponds to each type of targeting signal. It binds only to the correct targeting signal sequence. This second part of the system corresponds to the street addresses on buildings in a city.

Protein Targeting

To nuclei

The nuclear localization signal (NLS) is a sequence of several amino acids, Pro-Pro-Lys-Lys-Lys-Arg-Lys -Val-, containing a run of basic amino acids that is located within a protein sequence, rather than at the ends.
• This sequence is exposed somewhere on the surface of the protein. 
• It combines with a nuclear import receptor protein.
• The complex of the nuclear import receptor and the protein to be imported then binds to the fibrils that extend on the cytosol side of the nuclear pore complex. This interaction is specific and depends on the nuclear import receptor being bound to the NLS protein.
• The nuclear pore apparatus then transfers the nuclear import receptor-NLS protein complex into the nucleus. This step requires energy (GTP).
• The nuclear import receptor then detaches from the NLS protein, and the nuclear import receptor proteins return to the cytosol and are reused.

Figure 14-9 Import of proteins into the nucleus. Notice the role of the nuclear import receptor protein (blue). Link to animation

To or across membranes

Proteins targeted to or across membranes typically carry a signal sequence at the N terminal end of the protein.

To Mitochondria, Chloroplasts or Peroxisomes

Key concept: The protein containing the proper signal sequence binds to a receptor , bound. It is then unfolded as it traverses the membrane through a protein channel, and is refolded on the organellar side of the membrane. See Fig 14-10 (left). This requires a set of special organellar chaperone proteins.

• For mitochondria the signal sequence is: ⁺H₃N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-
  Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu- 
• The protein containing the signal sequence is synthesized in the cytoplasm.
• Signal sequence binds to a receptor in the organelle membrane
• Receptor - protein complex diffuses within membrane to a contact site.
• Protein is unfolded, moved across the membrane, and refolded. These operations are carried out by the protein transporter complex and its associated chaperone proteins. Remember chaperones from earlier discussion of protein processing? The signal sequence is the first part of the protein to enter the organelle.
• Once inside, the signal sequence is cleaved off by a specific peptidase.

To endoplasmic reticulum.

Synopsis. Synthesis of proteins entering the endoplasmic reticulum is initiated on free ribosomes. A targeting sequence of  hydrophobic amino acids near the amino terminal end of the growing polypeptide results in the binding of the ribosome to ER membrane and in insertion of the polypeptide into the endoplasmic reticuluum.

Proteins going to Golgi, endosomes, lysosomes and ER all enter the ER and don't come out again.

There are two groups of proteins targeted to the ER:

• Proteins that are completely translocated into the endoplasmic reticuluum. These proteins are soluble (not membrane proteins) and are destined for secretion, or for the lumen of another organelle. In all of these cases the proteins are  never part of membranes.
• Proteins that are inserted into membranes, and hence are only partially translocated into the endoplasmic reticuluum. These proteins may be destined for ER, another organelle, or the plasma membrane. In all of these cases the proteins stay within the membrane (e.g. cellulose synthase).

Let's deal first with the case of proteins that will be inserted into the ER lumen:

• The signal sequence is located at the N terminal end of the protein: ⁺H₃N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln-
• The long sequence of about 10 hydrophobic amino acid residues (in blue, above) is a membrane crossing domain.
• Targeting to the endoplasmic reticulum takes place through the interaction of the signal peptide sequence ( a sequence of at least eight hydrophobic amino acids at the amino terminal end of the polypeptide. The emerging signal sequence combines with a 'signal recognition particle' (SRP). This greatly reduces the rate of translocation and allows the ribosome to attach to the endoplasm reticulum by means of a special SRP receptor in the ER membrane.
• The ribosome becomes attached to a ribosome receptor that also functions as the translocation channel for the newly synthesized polypeptide. As the ribosome becomes attached, the SRP is removed and translation resumes. Figure 14-13. shows two components:
  • There is a Signal Recognition Particle (SRP) in the cytosol. This binds to the ER Signal sequence when it is exposed on the ribosome and slows protein synthesis long enough to allow the SRP to find the second part, the SRP Receptor.
  • The Signal Recognition Particle Receptor (SRPR) which is embedded in the ER membrane. We now have the new polypeptide synthesizing system in place and protein synthesis speeds up. It seems that the Signal Sequence opens the translocation channel.
• After translation is complete, the signal sequence, which is embedded into the ER membrane, is cleaved off of the protein by a specific signal peptidase, an enzyme that is present in the ER lumen. This leave the newly synthesized protein free in the lumen of the ER. See animation of this process

Proteins inserted into membranes:

• There are three types of hydrophobic signals used in insertion of membrane proteins. All of these are membrane crossing domains:
  • Signal peptide sequence - a cluster of about 8 -10 hydrophobic amino acids at the N-terminal end of a protein. This sequence remains in the membrane and is cleaved off of the  protein after transfer through the membrane. This is the same as the signal peptide sequence mentioned above.
  • Start transfer sequence. Similar to a signal sequence, but located internally (not at the N terminal end of the protein). It also binds to the SRP and initiates transfer. Unlike the N-terminal signal sequence, it is not cleaved after transfer of the protein.
  • Stop transfer signal. This is also a sequence of about 8 hydrophobic amino acid residues. It follows either a N-terminal signal sequence or a start transfer sequence. 
    • The stop transfer signal is a membrane crossing domain. It remains in the membrane. 
    • When it is is encountered the translocation channel is disassembled. 
    • The peptide is not cleaved.
    • Translation continues in the cytoplasm.  
    • If a subsequent Start Transfer sequence is encountered in the protein, a second SRP binds to the start transfer sequence and a new translocation channel is opened in the membrane.

The process works as follows:

Whenever a N terminal signal sequence or a start transfer sequence is produced by the translation process, it bind to a signal recognition particle that results in attachment to the ER and the formation of a translocation channel. If a stop transfer sequence is encountered, translocation is stopped, although translation continues to the end of the molecule. If a subsequent start transfer signal is encountered a new SRP binds and a new translocation channel is formed.

This diagram shows the relation between translocation control sequences (signal sequences, start transfer sequences, stop transfer sequences) and the arrangement of the protein in the membrane. How would the translocation control sequences have to be arranged to get the N terminal end of  the protein on the cytoplasmic side? - to get the C terminal end of the protein on the cytoplasmic side? Animations for insertion of membrane proteins (including multi-pass proteins).

Figure 14-15. Click to enlarge Animation of this process

Import of a membrane protein into the membrane of the ER. The blue sheath-like component shown in the figure is the translocation channel that moves the protein through the membrane. Notice that the Stop Transfer sequence (orange) results in the disassembly of the translocation channel. Note that the signal sequence at the N terminal end of the protein is cleaved off, releasing the N terminal portion of the protein into the ER lumen.  This example is a single pass membrane protein that contains a single membrane crossing domain (the stop transfer signal).

Vesicle Formation and Targeting

Fig. 14-17 Vesicle traffic in cells. Click to enlarge.

Individual protein molecules are targeted to various destinations within the cell. Individual proteins are not the only things moving in cells.  There is a tremendous flux of vesicles within most cell types. Vesicles form from the endoplasmic reticulum, the Golgi apparatus and the plasma membrane. They are used to transport membrane and proteins between many different membranous organelles.  Here we will be looking at how vesicles are formed and how they find their targets.

Vesicle formation and vesicle transport

Transport between these compartments takes place via vesicles. Vesicle transport is the means by which membrane transport occurs between compartments within the cell. Membrane proteins and soluble proteins contained within the vesicles are also transported.

For example, once the proteins are in the ER, they are transported by vesicles which bud  off  of the ER and fuse with the membrane of the target compartment. 

Vesicle transport presents substantial targeting problems: each vesicle must take correct cargo to correct target.

The key to this form of transport  lies in the vesicle coats.

There are several types of coats but all have two functions:

• to shape the membrane into a bud
• to capture molecules for onward transport. See animation.

Each bud has a distinctive coat protein on cytosol surface.

• The coat protein must shape the membrane into a bud.
• The bud must capture the correct molecules for outward transport
• After budding, the protein coat is lost.
• Bud can now interact with target - target interaction signals are now exposed.

Two examples of target protein systems are COP-coated vesicles involved in transport of vesicles from ER to Golgi and within Golgi.  and clathrin coated vesicles that carry proteins from the Golgi to endosomes or from the plasma membrane to endosomes.

The best studied vesicles coated by proteins are coated with a set of proteins called clathrins.

• Clathrin coated vesicles bud from the outer (trans) face of the Golgi complex or from the plasma membrane..
• Clathrin coated vesicles are involved in transport of materials by vesicles from the Golgi to the plasma membrane (exocytosis), as well as transport from the plasma membrane to endosomes (endocytosis) .

To form a bud and initiate the budding process you need to have stuff (cargo) in the package (vesicle) and then have to pinch it off.

Figure 14-19. Formation of coated vesicles.	This figure shows the process of clathrin coated vesicle formation at the cell surface. The same process occurs at the trans-Golgi to form vesicles that move toward the plasma membrane. See animation.
Fig 14-18 Click to enlarge.	Formation of a clathrin coated vesicle. Notice the thickness of the cargo material attached to the cargo receptors that extend through the membrane. The clathrins form a layer in the cytosol side of the membrane, and are very important for selection of cargo protein for transport. See animation.

This process requires the interaction of several components: cargo receptor, adaptin, clathrin and dynamin.

• The cargo molecule is picked up by the cargo receptor, which is an integral membrane protein.
• The cargo receptor/cargo complex is recognized by adaptin which combines with the cytosolic side of the cargo receptor molecule.
• The cargo/ cargo receptor/ adaptin complex then combines with clathrin on the cytosolic surface.
• Clathrin forms the curved bud membrane configuration
• Dynamin constricts the neck of the bud (vesicle), which then pinches off.
• Uncoating then occurs as the clathrin and adaptin are released and recycled.
• Each vesicle also has a specific targeting signal as described below.

The vesicle is now ready for transport.

Vesicle targeting

Over short distances, movement of vesicles is by diffusion. Transport of vesicles over longer distances is dependent on cytoskeleton-based motor proteins.

Docking must be specific (don't know how it works). For example, hemicellulose going to the plant cell wall is delivered to sites where cellulose synthesis is occurring. Complementary fit is part of the story, but snares are also involved.

Figure 14-20. Snares and specificity of vesicle transport.	Snares are proteins that result is specific attachment of vesicles to their target membranes. Snares occur as complementary pairs of proteins. The vesicle-snare (v-snare) is incorporated into the vesicle membrane, and the target-snare  (t-snare) is incorporated into the target membrane. Docking occurs by interaction of the v-snare and t-snare proteins. This binding is very specific.
Figure 14-21. Transport vesicle fusion.	Once the vesicle and the target membranes are docked, several other proteins join to form a 'fusion complex' that results in the fusion of the vesicle with the target membrane. Fig 4-21. Following the docking of a transport vesicle at its target membrane, a complex of membrane fusion proteins assembles at the docking site and catalyses the fusion of the vesicle with the target membrane.