Question

Question 1. Cystic Fibrosis (CF) is an example of a protein mis-folding disorder since most of the mutations associated with CF lead to the improper folding and processing of the plasma membrane-localized Cystic Fibrosis Transmembrane Conductance Regulator ion channel (CFTR).

The CFTR protein initially enters the endomembrane system through the process of co-translational translocation into the endoplasmic reticulum (ER) and is inserted as a Type IV membrane protein. N-linked glycosylation of the CFTR protein then takes place in the ER and continues in the Golgi.

Name the four types of sequence signals in the CFTR protein required for its proper targeting and processing up to this point (the Golgi) and give an explanation for each.

Answer 1

After translation process in ribosomes, the polypeptide chain generated undergoes several post-translational modifications and changes and are translocated to other area of the cell or secreted out of the cell, by the process of protein trafficking.

The proteins are recognized by a short signal sequence (6-12 amino acid sequence) present at the N-terminus, and are targeted to specific organelles, including endoplasmic reticulum, Golgi apparatus, lysosome, by protein targeting.

While some proteins are not associated with the signal peptide.

a. Lysosome targeting of protein occurs in Cis- Golgi network (CGN).

• N-Acetyl glucosamine (NAG) phosphorylases, first binds to Mannose residues.
• The NAG part is then removed, and Mannose -6- phosphate (M-6-P) is retained on the N-linked oligosaccharide.
• Mannose phosphorylation of proteins is the indication of protein transportation to lysosome.
• Phosphorylated protein is recognized by M-6-P receptor on trans- Golgi network (TGN), and these proteins are directed to lysosome. Signal patch on lysosome recognizes the M-6-P related protein.

b. Proteins are transported through protein coated membrane vesicles, like clathrin-coated, COPI coated, COPII coated. Some protein-cargos may be returned to the E.R, because of the presence of specific signal sequences, that interact with COPI or some other associated proteins.

• If the cargo-protein needs to be retained back to the E.R, it need specific sequence called retrieval signals to be added to their C-terminals.
• The E.R membrane proteins also contain these signal sequence at the C-terminal ends.
• The sequence for E.R resident protein is Lys-Lys-XX, and for soluble E.R protein is Lys—Asp-Glu-Leu and are designated for receptors called KDEL receptors or multi-pass trans-membrane protein. They can interact with COPI.
• Thus, addition or modification of C-terminus of the protein to generate a KDEL signal sequence will retrieve or retain the cargo-protein to E. R. instead of lysosome.

c. Nuclear localization sequence (NLS) allows the protein to be transported to the nucleus.

• NLS sequences are short sequences of about 6 to 8 amino acids, with arginine and lysine amino acids (basic or positive charged). These sequences can associate with nuclear transport receptors (TRs) and helps in transport of cytosolic protein through nuclear membrane pores.
• NLS sequences should be located with the protein, as internal sequences, and not as terminal sequences. Thus, the transport to nucleus may be inhibited, due to adding NLS at C-terminus.
• Proteins or polypeptides associated with the signal peptides: These are transported to nucleus, mitochondria or chloroplast.
• These peptides are usually translated in ribosomes associated with endoplasmic reticulum (RER).
• They contain N-terminal signal peptide.
• The signal sequence also contain more sequences like Start and stop sequences, spanning regions, GPI anchoring regions.
• For targeting to mitochondria, proteins contain N-terminal pre-sequence or matrix targeting sequence.
• This is composed of alternating charged and hydrophobic amino acid residues.
• These amino acids form an amphipathic helix that can target protein translocation across mitochondrial membranes and into the mitochondrial matrix.

d. Proteins or polypeptides without the signal peptides:

• These peptides are synthesized in free ribosomes.
• Proteins to be targeted to mitochondria are modified to have a presequence comprising of hydrophilic amino acid and charged residues.
• The protein acquires a structure of ampipathic helix and pass through mitochondrial membrane. Further, sub-compartment localization occurs as needed.