Protein Transport Through the Golgi

Protein Glycosylation in the Golgi

Glycosylation reactions in the Golgi occur in the cis-, medial-, and the trans-Golgi.

- As a protein moves through the Golgi, sugar residues are added and removed to produce a typical N-linked complex oligosaccharide [8]. These reactions are catalysed by different enzyme located in the cis-, medial-, and trans-Golgi e.g. galactosyltransferase and sialyltransferase are both found in the trans-Golgi [9].

- There are different variations in N-linked oligosaccharide structures that can occur due to differences in processing such as limited accessibility to certain parts of the N-linked oligosaccharide for relevant enzymes to act upon and therefore no further modifications will occur due to the substrate for the next enzyme in the pathway not being generated [9]. These proteins contain a high-mannose oligosaccharide as opposed to the complex oligosaccharide yielded from the complete pathway [9].

Figure 1.  Processing of glycoproteins in cis-, medial- and trans-Golgi to yield N-linked oligosaccharides. The enzymes catalysing each step are localized to the compartments they are indicated to. During Step 1, three mannose residues are removed, Step 3 two more mannose residues are removed. At Step 2 and Step 4, three GlcNAc residues are added, after this a single fucose is added which is Step 5. To complete processing, in Step 6, three galactose residues are added and finally, linkage of an N-acetylneuraminic acid residue to each galactose residue.

One function of some N-linked oligosaccharides is to prevent secretion of lysosomes by targeting lysosomal enzymes to them. In the cis-Golgi there are two reactions that take place which causes one or more mannose residues in resulting oligosaccharides to become phosphorylated [9]. The first reaction causes addition of a N-acetylglucosamine phosphate residue is added to the 6-carbon atom in a mannose in the N-linked oligosaccharide by N-acetylglucosamine phosphotransferase, an enzyme specific for lysosomal enzymes [9]. In the second reaction, the N-acetylglucosamine residue is removed by phosphodiesterase resulting in a mannose 6-phosphate residue [9].

Figure 2. In reaction 1, a GlcNAc phosphotransferase in the cis-Golgi transfers a acetylglucosamine phosphate group to the C6 of either one or more mannose residues. There is a recognition site on this enzyme that has signal segments bind to it (red) which is only present in cathepsin D and other lysosomal enzymes. Sugar residues that are modified by phosphotransferase are a decent distance from the signal segments which shows that the catalytic site and recognition site of the enzyme are distinct. Reaction 2 shows a phosphodiesterase removing the GlcNAc which leaves a phosphorylated mannose residue on the lysosomal enzyme.

Protein Sorting and Export from the Golgi

Proteins are transported through the secretory pathway from the Golgi to their final destinations.

- The pathway involved sorting proteins into different transport vesicles that bud off of the trans-Golgi network and the proteins are then delivered to their destined locations [8]. Some proteins are transported to the cell surface through a pathway of regulated secretion. Some proteins are carried to the membrane by a constitutive secretory pathway that accounts for incorporation of other proteins and lipids to the membrane along with continuous protein secretion [2]. Sorting of proteins into pathways occurs in the trans-Golgi network where packaging into specialized secretory vesicles occurs. These vesicles store the contents until a specific signal is recognised which tells the vesicle to fuse with the membrane.

Figure 3. Export from Golgi. Protein sorting occurs in the trans-Golgi and transported in vesicles that bud off and move to their final destinations such as lysosomes, constitutive secretion or regulated secretion [2]

• Proteins from the Golgi are packaged into vesicles that bud off of the Golgi and are either transported to lysosomes for digestion [1], transported for regulated secretion which sees proteins secreted in response to environmental signals such as release of hormones from endocrine cells or transported for constitutive secretion which leads to continual, unregulated secretion of proteins [2].

There are two models that have been proposed for how cargo moves through the Golgi but it is unknown.

The 'Stationary cisternae/vesicular transport' model

• This model suggests that each cisternae of the Golgi is a stable entity and secretory cargo is transported from one cisternae to another via vesicles moving along [6]. The Golgi is seen as a set of compartments. Each compartment contains a set of Golgi proteins, unique to that compartment. Secretory cargo that is newly synthesized is delivered to the cis-Golgi in COPII-coated vesicles and then moved from compartment to compartment in COPI-coated vesicles [6].
  

Figure 4. Vesicular transport between stable compartments. Secretory cargo travels from the ER to an intermediate compartment and cis-Golgi in different carries. Cargo moves across the stack through COPI vesicles that bud from a compartment and fuse to the next.

The 'cisternal maturation' model

• This model suggests that the cis-cisternae forms de novo by fusion of the membrane and moves down the stack towards the trans face like a conveyor belt, maturing in medial and trans cisternae on the way [5]. The cisternae will then disintegrate into secretory vesicles and other carrier types. COPI vesicles would then recycle resident Golgi proteins from older to younger cisternae. This could explain the biochemical polarity of the Golgi. A recent proposal was that the different compartments of the Golgi represent different kinetic stages of maturation. Transition from one stage to the next was postulated to be regulated by Rab GTPases [6].

Figure 5. Secretory cargo exits ER in different carries. These coalesce with eachother and COPI vesicles to form the intermediate compartment, which forms a new cisternae. Through COPI-mediated recycling of Golgi proteins from old cisternae, new cisternae matures. During this recycling, both small and large cargoes are carried through the stack. Finally the cisternae breaks down into anterograde and retrograde transport carriers.

COPI Vesicles

• COPI is a protein complex which coats trafficking vesicles which are trafficked from the cis Golgi to the trans Golgi as well as from the cis Golgi to the endoplasmic reticulum (1). COPI consists of 7 subunits which form a heteroheptameric protein complex (1). The roles of COPI vesicles include: enabling the formation of vesicles and aiding in the selection of protein and lipid cargo to be packaged (2).
  
• COPI vesicles contain a variety of different structures to enable efficient trafficking. Soluble cargo is captured transmembrane adaptor proteins (TRAPs), preventing any further interaction between the cargo and the vesicular membrane (2). Furthermore, the COPI vesicles also contain KDEL receptors which are capable of recognising ER proteins through the detection of the code presented at the c-terminus of these proteins (2).
  
• Despite the main role COPI vesicles within the secretion pathway is retrograde transport from the cis to trans Golgi, COPI vesicles also play a role in the transport of cargo from the endoplasmic reticulum to the Golgi. Despite the initial trafficking from the endoplasmic reticulum being performed by COPII vesicles, further transport is performed by tubulovesicular intermediates which are associated to COPI vesicles (2).