Question

9. Explain what two types of proteins are transferred from the cytosol to the ER. How are these proteins translocated into the RER? What are some examples of proteins that would fit into each of these two categories?

10. Explain the secretory and endocytic pathways.

11. Explain vesicle budding and protein coats. 12. Explain the basic role of SNARES in vesicle docking.

13. Explain the types of covalent modifications of proteins in the ER and Golgi. Relate these modifications to concepts we have discussed, i.e. di-sulfide bonds.

14. Explain the constitutive and regulated pathways from the Golgi. Include some examples of proteins that would use each pathway.

15. Explain the basics of receptor-mediated endocytosis.

16. Explain the mannose-6-phosphate tag for the enzyme delivery to lysosomes.

Answer 1

Q)9) There are two types of proteins which are transferred from cytosol to ER they are water soluble proteins and Prospective transmembrane proteins.

i) Water soluble proteins , these are heading either for secretion or for the lumen of an organelle, they are fully translocated across a ER.

ii) Trans membrane proteins are supposed to be in membrane of ER or in other cellular organelles or the Plasma membrane , they are partly translocated across ER.

Translocation of proteins across the membrane of the endoplasmic reticulum (ER) occurs by one of the two ways- they are cotranslationally, in this way translocation occurs at the same time, along with peptide synthesis by the ribosome, or posttranslationally, in it protein is first synthesized in the cytosol and then transported into the ER. Both the process of translocation are mediated by the same protein channel, it is Sec61 in eukaryotes and SecY in prokaryotes and archaea.

Cotranslational translocation-

In this proteins which are supposed to get translocated across the ER membrane have a distinct amino-terminal signal sequence, it is recognized by the signal recognition particle (SRP). In eukaryotes SRP is a large ribonucleoprotein , when it is bound to the ribosome and the signal sequence of the nascent peptide,it is able to stop protein translation by blocking entry of tRNA .

The ribosome is targeted to the ER membrane by a series of interactions, it starts with the binding of the SRP by the SRP receptor. The signal sequence of the nascent peptide chain then transferred to the protein channel, Sec61. By the binding of SRP to its receptor SRP gets dissociated from the ribosome, and the SRP and SRP receptor also gets dissociated from each other following GTP hydrolysis. As SRP and SRP receptor dissociate from the ribsome, and the ribosome can directly bind to Sec61.

The Sec61 translocation channel (known as SecY in prokaryotes) is a highly conserved heterotrimeric complex made up of , and subunits. The pore of the channel, formed by the -subunit, is blocked by a short helical segment which gets unstructured at the beginning of protein translocation, allowing the peptide to pass through the channel, the signal sequence of the nascent peptide gets inserted into the walls of the channel, by a side opening known as the lateral gate. During translocation, the signal sequence gets cleaved by a signal peptide peptidase, as a result amino terminus of the growing peptide gets detached, and remains free in the lumen of ER.

Posttranslational translocation in eukaryotes-

.During cotranslational translocation, the ribosome pushes the growing peptides into the ER lumen. During posttranslational translocation, additional proteins are required to ensure that the peptides should move unidirectionally into the ER membrane. Eukaryotes and prokaryotes have different mechanisms regarding the successful translocation of synthesized peptides.

In eukaryotes, post translational translocation requires the Sec62/Sec63 complex and the chaperone protein BiP, BiP is a member of the Hsp70 family of ATPases, it is a group characterized by having a N-terminal nucleotide-binding domain (NBD), and a C-terminal substrate-binding domain (SBD) which binds to peptides. The nucleotide binding state of the NBD determines whether the SBD can bind to a substrate peptide. When NBD is bound to ATP, the SBD is in open state, it allows peptide release, when NBD bound to ADP, the SBD is in closed state and peptide-bound.

The membrane protein complex Sec62/Sec63 helps in activating the ATPase activity of BiP via a J-domain located on the lumen-facing portion of Sec63. The SBD of BiP binds non-specifically to the peptide, when it enters ER lumen, and keeps the peptide from sliding backwards .

Posttranslational translocation in prokaryotes-

In prokaryotes, translated peptides are actively pushed by the SecY channel by the SecA protein. SecA is made up of a nucleotide-binding domain, a polypeptide crosslinking domain, and helical wing and scaffold domains .

At the time of translocation, a region of the helical scaffold domain forms a two-finger helix, which inserts into the cytoplasmic side of the SecY channel, and pushes the translocating peptide . A tyrosine present at the tip of the two-finger helix plays a critical role in translocation, and it is thought to make direct contact with the translocating peptide.

The polypeptide crosslinking domain (PPXD) forms a clamp , it is thought to open when translocating peptide is pushed into the SecY channel by the two-finger helix, and close when the two-finger helix resets to its "up" position.

The conformational changes of SecA occurs by it's nuclease activity, by hydrolysis of one ATP in each cycle.

Q)10) Explain secretory and endocytic pathway.

Secretory pathway - Proteins which are supposed to get secreted follow the secretory pathway, in it the cell secretes proteins into the extracellular environment. This pathway also processes membrane-bound proteins (whether in the cellular membrane or in the ER or Golgi membranes ).The endoplasmic reticulum is the first step in the secretory pathway. Its membrane is continuous with the outer nuclear membrane,

In this proteins and lipids are synthesized in the ER and then transported to the Golgi. Proteins are sorted in the Golgi and sent to the plasma membrane, lysosome or secretory vesicles. Transport of protein and lipid between membrane-bound compartments is mediated by vesicles they bud from one compartment and fuse with the subsequent compartment. In this cells maintain the integrity and functionality of the ER and Golgi by inhibiting resident proteins from entering vesicles and retrieving the proteins which escape.

The endocytic pathway in mammalian cells consists of distinct membrane compartments, they internalize molecules from the plasma membrane and recycle them back to the surface or sort them for degradation.

The different components of endocytic pathway are - Early endosomes, Late endosomes, Lysosomes.

Early endosomes- These are the first compartment of the endocytic pathway.they are found in the periphery of the cell, and they receive the vesicles coming from the cell surface. They have a typical tubulo-vesicular structure and have a mildly acidic pH. They are mainly the sorting organelles. Here many endocytosed ligands dissociate from their receptors in the acid pH of the compartment, and from here many receptors recycle back to the cell surface (via tubules). It is also the site of sorting into transcytotic pathway to later compartments (like late endosomes or lysosomes) via transvesicular compartments (like multivesicular bodies (MVB) or endosomal carrier vesicles (ECVs)).

Late endosomes- They receive endocytosed material going on the way to lysosomes, usually from early endosomes in the endocytic pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. Late endosomes usually contains proteins characteristic of lysosomes, having lysosomal membrane glycoproteins and acid hydrolases. They are acidic (approx. pH 5.5), and are also part of the trafficking pathway of mannose-6-phosphate receptors. Late endosomes are supposed to mediate a final sorting process before the delivery of materials to lysosomes.

Lysosomes- These are the last compartment of the endocytic pathway. Their break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds. These are then returned back to the cytoplasm as new cell-building materials. For this process, lysosomes use aroun 40 different types of hydrolytic enzymes, all these gets produced in the endoplasmic reticulum, modified in the Golgi apparatus and function in an acidic environment.The approximate pH of a lysosome is 4.8. They have lysosomal membrane proteins and active lysosomal hydrolases, but no mannose-6-phosphate receptor. They are usually the principal hydrolytic compartment of the cell.