Protein Processing II: Endomembrane System -Vesicle Transport and the Golgi Apparatus

In this discussion we will be dealing with the Endoplasmic Reticulum and the Golgi Apparatus. These compartments of the endomembrane system are linked by extensive vesicle traffic that moves proteins in both directions, from ER to Golgi (red arrow), and some proteins from Golgi to ER (blue arrow). This traffic of proteins, like that occurring throughout the rest of the endomembrane system occurs by means of vesicle transport. We start by looking at vesicle transport and then move on to discussion of the Golgi apparatus.

Key ideas:

• Vesicles form because of interaction between proteins inserted into the membrane and special coat forming proteins.
• Formation of the vesicle is an essential for concentration of cargo proteins.
• Vesicles are targeted by means of protein-protein interaction (address house number analogy).
• Golgi is the main site of protein processing and sorting for different destinations.

A. Vesicle Formation and Targeting

Fig. 14-17 .

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 look at how vesicles are formed and how they find their targets.

Vesicle formation and vesicle transport

• Transport between compartments takes place via vesicles.
• Membranes, with both proteins and lipids, and the soluble proteins contained within the vesicles are transported.
• For example, once the proteins are in the ER, they are transported by vesicles that bud  off of the ER and fuse with the membrane of the target compartment. 

There are two major problems:

• Formation of vesicles and selection of their contents and
• Targeting - each vesicle must take the correct cargo to proper target.

Formation of vesicles and selection of their contents

Vesicles form by budding from membranes of ER, Golgi and the plasma membrane. Micrograph.

Each bud has a distinctive coat protein on cytosol surface.

• The coat protein shapes the membrane into a bud.
• The bud captures the correct molecules for outward transport because cargo receptors are attached through the membrane to the coat protein complex. See animation.
• After budding, the protein coat is lost.
• Bud can now interact with target - target interaction signals are now exposed.

Figure 14-19.	Consider endocytosis at the plasma membrane. Figure 14-19 shows the process of vesicle formation. The same process occurs at the trans-Golgi to form vesicles that move toward the plasma membrane.
Fig 14-18	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.

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. For example, hemicellulose going to the plant cell wall is delivered to sites where cellulose synthesis is occurring. Part of this story involves snares.

Figure 14-20.

Snares are proteins that result in 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. This is the part of the targeting process that corresponds to address on the envelope and house number on the mail box in the postal delivery analogy.

Figure 14-21.

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. The key protein here is snap-25 that interacts with the target snare prior to joining of target and vesicle snares.

B. Protein Processing and the endomembrane system

All proteins are processed

After translation on ribosomes in the cytosolic compartment all proteins are processed either in the cytosol or in the ER/Golgi system. 

The initial stages of protein processing involve folding. 

• Remember that folding of proteins takes place through interaction with chaperone proteins (see pp 139-40 and 232, 468-9). 
• Proteins that are not properly folded are destroyed.  In the cytosol compartment they are tagged with ubiquitin and destroyed by proteasomes.

Modification of membrane proteins and proteins destined for secretion in the endoplasmic reticulum. Proteins targeted to the ER will end up as membrane proteins or as soluble proteins destined for vesicles (e.g. lysosomal proteins) or secretion. 

Other forms of processing occur in the ER lumen. 

• covalent modification (phosphorylation, methylation, acetylation and formation of disulfide bridges), and 
• cleavage of the initial protein product to produce a smaller active protein. Cleavage occurs commonly in the case of digestive enzymes and other secreted proteins (e.g. insulin).
• glycosylation - addition of polysaccharides to form glycoproteins.  

• The enzymes that carry out these reactions are located in the lumen of the E R, and not in the cytosol.
• This means that the proteins being glycosylated are bound for secretion  or are membrane proteins.  

Thought question: In the case of membrane proteins, what part of the protein would be glycosylated. The inside (cytosolic) part or the outside part?

Figure 14-22. Protein glycosylation in the ER. When polypeptide chains enter the endoplasmic reticulum they are immediately glycosylated by the addition of an oligosaccharide chain that is transferred as a single unit from a phospholipid called dolichol to an asparagine residue in the protein

Note in the figure above (14-22) that the oligosaccharides are added as an intact pre-fabricated unit consisting of 14 linked sugar residues transferred from a phospholipid anchor in the membrane.

The glycolipid units are 

• initially synthesized in the cytosol and embedded in the cytosolic face of the membrane. They are then
• flipped to the external (lumen) leaflet of the membrane. 
• joined covalently to asparagine in asn -X- (ser or thr) sequence tag.

C. Control of protein exit from the ER.

Some proteins are retained in the ER (for example, the enzymes that modify the oligosaccharides that are added to proteins)

• These proteins carry an ER retention signal (KDEL sequence) at their carboxyl ends. See Table 14-3.
• Even if they get out of the ER into vesicles they are brought back to the ER by retrograde (trans to cis) movement of transport vesicles. This is another example of protein targeting via an internal encoded target signal.

Proteins must be folded and processed properly.

• Proteins that are normally exported from the ER must be properly folded.  Abnormally proteins are retained by chaperone molecules and degraded if they do not cooperate and fold correctly.
• Many multi-polypeptide proteins, such as antibodies, are assembled in the ER. If these proteins are not properly assembled (via folding and the formation of disulfide bridges), the proteins are degraded.
• Cells make lots of mistakes in the assembly of proteins. They just do not let them be seen in public.

Proteins that get out of the ER are transferred to the Golgi apparatus by COPII-coated vesicles.

D. The Golgi Complex  

Fig 14-24

Study Figures 14-24 and 14-17 in text for basic structure of the organelle. The Golgi complex consists of stack of flattened sacs (cisternae) with expanded or swollen ends. The Golgi complex has two functionally and structurally different faces. The behaviour of the Golgi depends on the presence of other organelles, e.g. cytoskeleton for support and movement.

Overview of Golgi structure.

The Golgi consists of three components:

• the cis Golgi network
• the Golgi stack and 
• the trans Golgi network

Each Golgi stack has two faces,

• The cis face, near the ER, is the entry face that receives small membrane vesicles from the ER. The vesicle membranes are incorporated into the Golgi membranes and the contents of the vesicles enter the Golgi cisternae.
• The trans face, facing away from the nucleus toward the plasma membrane, is the exit face where vesicles leave the Golgi and move to their targets, including the exterior of the cell.

Here are some images of Golgi apparatus from the Biol 200 tutorial. Identify

• the cis and trans faces of golgi,
• the trans Golgi network, and
• transport vesicles in these pictures.

The Golgi cisternae contain a variety of transglycosylases ( enzymes that move sugars from one molecule to another) that modify the oligosaccharide chains of glycoproteins. Different enzymes reside in different regions of the complex.

The gruesome details of Glycosylation in the Golgi Complex.

• How are these enzymes kept in place and
• how is the flow of target proteins through the Golgi regulated?

Golgi dynamics:

The flow of cargo proteins through Golgi apparatus is from cis to trans.   (ER > transitional vesicles > cis Golgi Network > cis cisterna > medial cisterna > trans cisterna > trans Golgi network > secretory vesicles).

Despite this flow there are many resident proteins that are localized in particular parts of the Golgi. Two classes of models have been presented to explain the cis to trans flow of cargo proteins while the resident proteins stay in place.

1. Vesicle transport model:

• Cargo proteins (but not resident proteins) are moved from stack to stack by vesicle transport. 
• This sorting also involves both
  • forward, anterograde (cis to trans), and
  • backward, or retrograde (trans to cis), flow of vesicles with proteins, moving back up the stack.
• Resident proteins that are carried to locations trans to their normal location are transported back by retrograde movement of vesicles

2. Cisternal maturation model:

• The cis-most cisterna is the youngest, having been recently formed from incoming vesicles
• The trans-most cisterna is the oldest and breaks up into vesicles as material is moved to the trans Golgi network
• Cargo proteins are carried with the cisterna; resident proteins are returned to their proper location by retrograde movement of vesicles.

There is evidence for both  processes, and the extent to which the actual situation conforms to one model or the other varies among cell types.

Vesicles from the trans face of the Golgi stack enter the trans Golgi network, that acts as a sorting and distribution centre.

Vesicles leave the Golgi for a number of destinations. These include: 

• inclusion in lysosomes (for example, enzymes involved in intracellular digestion), 
• incorporation into dense core secretory vesicles that are stored and later released through the regulated secretory pathway (example, digestive enzymes in the pancreas) and
• vesicles containing membrane and proteins that are immediately released to the surface via the constitutive  secretory pathway (example, cell coat proteins).