This mechanism makes finding the thermodynamically optimal conformation easier by preventing some potential suboptimal conformations from forming. These chaperone proteins bind to new proteins as they enter the lumen through the translocon. In addition to simply preventing incorrect bonds that would have to be broken, they also prevent premature interaction of multiple polypeptides with one another. This problem can occur when immature polypeptides have not undergone proper folding that would usually hide their interaction domains within the protein; exposed interaction domains can lead to indiscriminate binding and potentially precipitating insoluble protein aggregates.

Protein Degradation and Quality Control

Improperly folded proteins can contribute to many disease processes related to misfolding events. Typically, folding is facilitated in the ER using a chaperone called binding immunoglobulin protein; however, an altered protein (due to mutation) can lead to aggregation. Binding immunoglobulin protein requires an HSP70 molecular chaperone in the RER lumen that binds newly synthesized proteins as they are cotranslated and prevents premature folding. Binding immunoglobulin protein accumulation can initiate the unfolded protein response (Figure 7).

 

E3 ubiquitin ligase is often responsible for tagging aggregates using a small protein called ubiquitin, which targets the protein to the proteasome. The proteasome consists of two subunits (19S and 20S) to make a functional 26S proteasome. Inside the proteasome, the polypeptide chains are cleaved back to their native amino acids for potential reuse in other translational events. However, if the aggregates accumulate, in some instances, they can contribute to any number of neurodegenerative disorders.

The Golgi Apparatus

As mentioned previously, vesicles can bud from the ER, but where do the vesicles go? Before reaching their final destinations, the lipids or proteins within the transport vesicles must be sorted, packaged, and tagged so they end up in the right place. Sorting, tagging, packaging, and distributing lipids and proteins occurs in the Golgi apparatus (also called the Golgi body), a series of flattened membranous sacs stacked together. Each membranous sac has a single lipid bilayer and is called a dictyosome (Figure 8). The Golgi apparatus was discovered within the endomembrane system in 1898 by Italian scientist Camillo Golgi (1843–1926), who developed a novel staining technique that showed stacked membrane structures within the cells of Plasmodium, the causative agent of malaria.

 

The Golgi apparatus has a receiving face, called the cis-face, near the ER and a releasing face, called the trans face, on the side facing away from the ER, toward the cell membrane (Figure 9). The transport vesicles that form from the ER travel to the receiving face to fuse with it and empty their contents into the lumen of the Golgi apparatus. As the proteins and lipids travel through the Golgi, they undergo further posttranslational modifications. The most frequent modification is adding short sugar molecule chains (glycosylation). Additionally, some proteins require Golgi-associated cleavage to produce a mature protein ready for trafficking. The Golgi packages the modified and tagged proteins into vesicles that bud from the trans face of the Golgi. Some of these vesicles, called transport vesicles, deposit their contents into other parts of the cell for use there. Other vesicles, called secretory vesicles, fuse with the plasma membrane and release their contents outside the cell.

 

Glycosylating enzymes in the Golgi apparatus modify lipids and proteins to produce glycolipids, glycoproteins, or proteoglycans. Glycolipids and glycoproteins are often inserted into the plasma membrane and are crucial for signal recognition by other cells or infectious particles. Distinguishing different types of cells from one another can be done by looking at the structure and arrangement of the glycolipids and glycoproteins in their plasma membranes. These glycolipids and glycoproteins commonly also serve as cell surface receptors.

The amount of Golgi in different cell types again illustrates that form follows function within cells. Cells that engage in a great deal of secretory activity (such as cells of the salivary glands that secrete digestive enzymes or cells of the immune system that secrete antibodies) have relatively more Golgi. In plant cells, the Golgi has an additional role of synthesizing polysaccharides, some of which the cell wall incorporates and some of which other parts of the cell use.

Roles of the ER and Golgi Apparatus in Protein Glycosylation

Glycosylation is the reaction in which an oligosaccharide (or glycan) attaches to another molecule’s hydroxyl or other functional group. In biology (but not always in chemistry), glycosylation usually refers to an enzyme-catalyzed reaction. Glycosylation is a form of cotranslational and posttranslational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. Proteins tend to clump or stick together (aggregate), and adding a sugar coating makes the polypeptide puffier or swollen (Figure 10). Aggregation influences folding and fills the spaces between molecules and entire cells, influencing cell-to-cell interactions with particular importance in cell adhesion. Glycosylated proteins are also important and clinically relevant opportunistic receptors for viruses and toxins entering the cells through receptor-mediated endocytosis. They are also common cell surface antigens (of which ABO blood types are probably the most known example). Lastly, added sugar residues often act as molecular flags or recognition signals to other cells that come in contact with them.

The following two general types of glycosylation reactions occur in cells:

• N-linked glycans attach to the nitrogen atom of the side chain amide group in asparagine residues. N-linked glycosylation requires a special lipid called dolichol phosphate to participate.
• O-linked glycans attach to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine or hydroxyproline side chains, or to oxygens on lipids such as ceramide.

N-Linked Protein Glycosylation Begins in the RER

N-linked glycosylation begins in the RER, but O-linked glycosylation does not occur until the polypeptide moves into the Golgi apparatus (Figure 11). Therefore, it is also the case that N-linked glycosylation usually begins as a cotranslational mechanism, whereas O-linked glycosylation must occur posttranslationally. Other key differences in the two types of glycosylation are as follows:

• N-linked glycosylation occurs on asparagine (Asn) residues within an Asn-X-Ser or Asn-X-Thr sequence (X is any amino acid other than Pro or Asp). O-linked glycosylation occurs on the side chain hydroxyl oxygen of either serine or threonine residues determined not by surrounding sequence but by secondary and tertiary structure.
• N-linked glycosylation begins with a “tree” of 14 specific sugar residues that are then pruned and remodelled but remain fairly large. O-linked glycosylation is a sequential addition of individual sugars that does not usually extend beyond a few residues.

The N-linked glycosylation process occurs in eukaryotes, widely in archaea, and very rarely in bacteria. The protein and the cell in which it is expressed determine the nature of N-linked glycans attached to a glycoprotein. It also varies across species. Different species synthesize different types of N-linked glycan.

N-linked glycan biosynthesis occurs via the following three main steps:

• Synthesizing a dolichol-linked precursor oligosaccharide
• En bloc transfer of the precursor oligosaccharide to a protein
• Processing the oligosaccharide

Precursor oligosaccharides undergo synthesis, en bloc transfer and initial trimming in the RER. The Golgi apparatus carries out the subsequent processing and modification of the oligosaccharide chain. Thus, glycoprotein synthesis is spatially separated in different cellular compartments. Furthermore, the type of N-glycan added to a protein depends on the different enzymes present in these cellular compartments. This is yet another example of cellular compartmentalization.

However, despite the diversity, all N-glycans are synthesized through a common pathway with a common core glycan structure. The core glycan structure consists of two N-acetylglucosamine and three mannose residues. Further elaboration and modification of this core glycan results in a diverse range of N-glycan structures.

Technically, N-glycosylation begins before any protein translation occurs, as the dolichol pyrophosphate oligosaccharide (i.e., the sugar “tree” – not an official term, by the way) is synthesized in the RER (Figure 12) without being triggered by translation or protein entry.

 

Dolichol is a long-chain hydrocarbon (14 to 24 isoprene units of 4+1 carbons) found primarily in the RER membrane. It serves as a temporary anchor for the N-glycosylation oligosaccharide during synthesis or while waiting for an appropriate protein to glycosylate. The oligosaccharide synthesis begins with adding two N-acetylglucosamine residues to the pyrophosphate linker, followed by a mannose. From this mannose, the oligosaccharide branches, with one branch receiving three more mannose residues and the other receiving one. So far, these additions to the oligosaccharide have been taking place in the cytoplasm. Now, the glycolipid is flipped inwards to the ER lumen. Once in the lumen, it obtains four more mannoses, and three glucose residues top off the structure.

The enzymes completing glycosylation are glycosyltransferases specific to both the added sugar residue and the target oligosaccharide. The sugars used by the enzymes are not simply the sugar but nucleotide sugars — usually a sugar linked to a nucleoside diphosphate(e.g., uracil diphosphate glucose (UDP-glucose) or GDP-mannose).

The N-linked oligosaccharide has two physiological roles:

• Acting as the base for further glycosylation
• Being used as a marker for error-checking of protein folding by the calnexin-calreticulin system (Figure 13)

Once the oligosaccharide attaches to the new polypeptide, further glycosylation begins when glucosidase I and glucosidase II remove two of the glucose. The last glucose is necessary to help the glycoprotein dock with either calnexin or calreticulin (Figure 13, Step 3). The chaperones calnexin and calreticulin have similar functions that help proteins fold and are associated with protein disulfide isomerase A3. Both chaperones temporarily hold onto the glycoprotein to give it time to (re)fold and possibly rearrange disulfide bonds. While calnexin is an integral protein embedded in the RER membrane, calreticulin is soluble in the RER lumen. Glucosidase II removes the glucose from the glycoprotein to dissociate the glycoprotein from calnexin/calreticulin. If the glycoprotein has not fully folded, the enzyme UDP-glucose:glycoprotein glucosyltransferase recognises it, adding back the glucose residue and forcing the gycoprotein to go through the calnexin/calreticulin cycle again in hopes of folding correctly. If it has folded correctly, a mannosidase can recognise it, which removes a mannose, completing the glycosylation modifications in the RER.

 

Most glycoproteins continue oligosaccharide remodelling once they have been moved from the RER to the Golgi apparatus by vesicular transport (Figure 14). There, various glycosidases and glycosyltransferases prune and add to the oligosaccharide. Although the glycosylation is consistent and stereotyped for a given protein, it is still unclear exactly how the glycosylation patterns are determined. In summary, N-glycan processing happens in RER and the Golgi body. Precursor molecules undergo initial trimming in the RER and subsequent processing in the Golgi. N-linked glycoproteins in the Golgi may take on additional sugar residues in the form of O-linked glycans.

 

O-Linked Protein Glycosylation Takes Place Entirely in the Golgi

O-linked glycoproteins begin their glycosylation when the Golgi-specific enzyme, N-acetylgalactosamine transferase, attaches an N-acetylgalactosamine to the hydroxyl group of a serine or threonine (Figure 15). Determining which residue to glycosylate appears to be directed by secondary and tertiary structures, and the process often occurs in dense clusters of glycosylation. Despite being relatively small additions (usually less than five residues), the combined oligosaccharide chains attached to an O-linked glycoprotein can contribute over 50% of the glycoprotein’s mass. Two well-known O-linked glycoproteins are mucin, a component of saliva, and ZP3, a component of the zona pellucida (which protects egg cells). These two examples also illustrate a crucial property of glycoproteins and glycolipids in general: sugars are highly hydrophilic and hold water molecules to them, greatly expanding the volume of the protein.

Glycosyltransferases and ABO blood groups

As mentioned above, glycosylated proteins and lipids are common cell surface antigens; these contribute to human ABO blood types (Figure 16).

 

Key Concepts and Summary

As discussed in Unit 3, Topic 1, newly synthesized proteins enter the biosynthetic-secretory pathway in the ER by crossing the ER membrane from the cytosol. During their subsequent transport — from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface and elsewhere — these proteins pass through a series of compartments, where they are successively modified. Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways. The pathway from the ER to the cell surface involves many sorting steps, which continually select membrane and soluble luminal proteins for packaging and transport — in vesicles or organelle fragments that bud from the ER and Golgi apparatus.

The endomembrane system includes the nuclear envelope, lysosomes, vesicles, the ER, the Golgi apparatus, and the plasma membrane. These cellular components work together to modify, package, tag, and transport proteins and lipids that form the membranes.

The RER modifies proteins and synthesizes phospholipids in cell membranes. The SER synthesizes carbohydrates, lipids, and steroid hormones, detoxifies medications and poisons, and stores calcium ions. Sorting, tagging, packaging, and distributing lipids and proteins take place in the Golgi apparatus. Budding RER and Golgi membranes create lysosomes. Lysosomes digest macromolecules, recycle worn-out organelles, and destroy pathogens.

Glycosylation is the process by which a carbohydrate covalently attaches to a target molecule, typically a protein and lipid. A glycosylated protein is called a glycoprotein, and a glycosylated lipid is called a glycolipid. This modification serves various functions. For instance, some proteins do not correctly fold unless they are glycosylated. In other cases, proteins are unstable unless they contain oligosaccharides linked at the amide nitrogen of specific asparagine residues.

N-linked glycosylation is a prevalent form of glycosylation important for folding many eukaryotic glycoproteins along with cell-to-cell and cell-to-extracellular matrix attachment. It begins in the RER. O-linked glycosylation is a form of glycosylation that occurs in eukaryotes in the Golgi apparatus but also in archaea and rarely in bacteria.¹

Key Terms

endomembrane system

group of organelles and membranes in eukaryotic cells that work together modifying, packaging, and transporting lipids and proteins

endoplasmic reticulum (ER)

series of interconnected membranous structures within eukaryotic cells that collectively modify proteins and synthesize lipids

Golgi apparatus

eukaryotic organelle comprised of a series of stacked membranes that sorts, tags, and packages lipids and proteins for distribution

lysosome

organelle in an animal cell that functions as the cell’s digestive component; it breaks down proteins, polysaccharides, lipids, nucleic acids, and even worn-out organelles

nuclear envelope

double-membrane structure that constitutes the nucleus’ outermost portion

rough endoplasmic reticulum (RER)

region of the endoplasmic reticulum that is studded with ribosomes and engages in protein modification and phospholipid synthesis

smooth endoplasmic reticulum (SER)

region of the endoplasmic reticulum that has few or no ribosomes on its cytoplasmic surface and synthesizes carbohydrates, lipids, and steroid hormones; detoxifies certain chemicals (like pesticides, preservatives, medications, and environmental pollutants), and stores calcium ions

vesicle

small, membrane-bound sac that functions in cellular storage and transport; its membrane is capable of fusing with the plasma membrane and the membranes of the endoplasmic reticulum and Golgi apparatus