Question

4. Ex plain how proteins produced in the rough endoplasmic reticulum can be secreted into the extracellular environment without ever crossing the plasma membrane. Use the terms rough endoplasmic reticulum ribosome, lumen, Golgi apparatus, cell membrane in your answer.

Answer 1

Protein production in the rough endoplasmic reticulum (RER):

Initially, mRNA transcription occurs in the nucleus. Further mRNA is going to be translated into more than 200 amino acid peptide on the ribosomes. This peptide is going to undergo various co-translational & post-translational protein- folding in “Endoplasmic reticulum lumen & Golgi apparatus” finally produces a mature protein. This is going to be transferred to vesicles where it undergoes 28 different types of collagen fibrils via Glycolysylation is the predominant process.

Posttranslational processing:

This is the processing of nascent protein in endoplasmic reticulum by adding polysaccharides into functional proteins after protein synthesis.

Normally, based upon the presence of granules, which are studded with ribosomes, it is of two types, rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER). The RER contains ribosomes and hence capable to synthesize polypeptides.

The proteins that are intended to be used within the cell (intracellular proteins) are produced by the ribosomes that float freely in the cytoplasm.

The proteins that are intended to be secreted out of the cell are synthesized by the ribosomes of the RER. This system contains “cisternae.” They are the fluid filled sacs, into which the synthesized proteins are released.

They modify these proteins by different processes. They enter into the endoplasmic reticulum as the lipid end of the protein (first 30 amino acids are hydrophobic) is attracted by the lipid component of the endoplasmic reticulum. Once they are in the ER, the hydrophobic chain is removed by the enzymatic process.

Glycosylation, ubiquinizaiton etc are co-translational and post-translational modifications of the nascent proteins in the cells.

For example, Glycosylation of a hormone affects the binding affinity to its cognate receptors because N-linked or O-linked glycolysation of “peptide hormone” acts as a ligand to bind to the receptor & this glycolysation is going to modulate the overall stability of hormone –receptor complex in a microenvironment. Recent research has illustrated that presence of “glycans or polysacchradie units linked to peptide hormones” has prolonged “Half-life” of this protein including hormones.

The secretory proteins are transported to the Golgi apparatus for further modification. For example, the glycosylation of the proteins by the addition of carbohydrates.

The proteins are differentiated based on their functions and destination by Golgi apparatus. These products are packaged in vesicles and transported into their destinations.

Degradation of useless proteins:

The proteins are differentiated based on their functions and destination. These products are packaged in vesicles and transported into their destinations.

Protein degradation is a part of protein regulation. Some of the proteins are degraded by the lysosomes. However, some of the proteins are also degraded outside the lysosomes by processes that require energy in the form of ATP.

During this process, the proteins to be degraded are tagged by the “ubiquitin” molecules, which are polypeptides of 76 amino acids length. These ubiquitin molecules bind to “lysine” of the target protein. Proteins tagged by the ubiquitin are then degraded by proteasomes.

Complete mechanisms of protein formation:

Protein folding with is associated with both hydrophobic and hydrophilic amino acids together to form strong covalent interaction through hydrogen bonds, Vanderwall's interactions, disulfide brides in opposite to conformational entropy. This is because the entropy values are reduced as protein is unfolded or at denatured state, therefore protein folding is spontaneous. Lowering of free energy is maximum when a protein is in aqueous solution with stretched out form. Therefore, when the protein is completely stretched -out or folded in an aqueous solution it is at an entropy minimum from lowest to greatest.  

Post-translational modifications:

Protein phosphorylation: addition of serine, threonine with a phosphate group to generate a modified protein

Sumoylation: it is characterized by the addition of tiny folded proteins with target other “target proteins” results in a change in function and stability

Ubiquitination: It is the degradation of native protein by tagging

For example, a tag can be an amino acid sequence or hydrophobic domain with an oligosaccharide (by glycosylation). This tag mainly used to enable the vesicles in delivering the material either by intracellular transport from endoplasmic reticulum or by extracellular transport.

Tag proteins are the protein, which grafted or incorporated into the recombinant proteins by using various biological methods. These tag proteins are predominantly useful in the protein structure and chemical modification or precise enzymatic alteration. The following are examples of the tag proteins.

Initially, glycan assembly is triggered outside ER i.e. in cytosol followed by glycan synthesis finally flipped into the lumen of ER via flippases. Glycolysylation is the predominant process, which is N-type, or O-type where the addition of polysaccharide chains done using appropriate enzyme specifically located in the lumen of the endoplasmic reticulum. Finally, the proteins being glycosylated bound then secreted as membrane glycoproteins with core oligosaccharides into the lumen of ER via flipping through flippases. These glycoproteins usually processed and transported to the plasma membrane via vesicular transport. For the proteins, which need to be fixed with any orientation in the lumen of the ER also associated with the same glycolysation.

Glycosaminoglycans are predominantly extracellular molecules and they are linked covalently with other protein moieties to produce proteoglycans, collagen, elastin laminin etc. Hyaluronic acid belongs to glycosaminoglycan and it is unsulphated. This is not going to link to a core protein directly and it is bound by the proteoglycans form bottle brush structure.

Protein translocation into the ER: Nascent secretory proteins are going to transport into the ER after “posttranslational & co-translational modification” via “molecular chaperones” that are targeted to the translocation machinery through protein translocation channels at the ER membrane. Membrane proteins often maintain the same as they adopted during their initial synthesis & the topology of a membrane protein is constantly conserved & preserved in which the “similar segments of protein face “cytosol”. Therefore, membrane proteins possess a unique orientation until they reach lipid bilayer through intracellular cargo by vesicular transport after biosynthesis on the ER membrane.

Chaperones role in protein folding: These are the components, which have meticulous “affinity” to promote adequate folding of an unfolded folded protein in their intermediate state finally allow to form a three-dimensional structure with higher stability in a slow & protected environment. The mechanism of recognition of chaperone of an unfolded protein is through “exposed hydrophobic residues” as this process utilizes ATP to promote appropriate stable folding thus chaperone reverse excess folding also.

1. Prenylation- Prenylated proteins: These are anchoring the lipid bilayer membrane of the cell by the prenylation finally form glycoproteins and glycolipids. There is an initial box called CAAX, to this box meticulously two components attached through covalent bonds. Those two components are farnesyl or geranylgernayl by the process of prenylation which can be indicated by c-cysteine as the initial site for prenylation.

2. Myristoylation -Fatty acylated proteins: These are lipid –protein anchored components predominantly formed by two processes such as N-myristoylation i.e. attachment of the fatty acid such as myristic acid through an amide bond to the terminal glycine linkage.

On the other hand, S-palmitoylation is another lipid anchoring process where palmitic fatty acid is added by acylation to the thioester linkage and cysteine bond.

3. Glycosylphosphatidylinositol-linked proteins: These are specific GPI complex lipid-anchored proteins considerably possess a variety of innervated components such as a phosphoethanolamine, and another component is phosphatidylinositol (sugar components).