Question

To examine the hierarchy of signal sequence targeting in a cell, you construct a variety of proteins containing different combinations of signal sequences. Based on your extensive knowledge of protein targeting, predict the location of each of these fusion proteins listed below. Provide the logic for your predictions.

(A) ER signal sequence and NLS

(B) mitochondrial signal sequence and KDEL sequence

(C) matrix-targeting sequence and a stop-transfer sequence

Answer 1

A)

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.

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)

• 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.