Question

1.Deletion of the KKXX sequence in the translocation would lead to it trafficking to?

2.Addition of a KDEL to a protein X that is normally secreted would lead to it being trafficked to?

3.Blocking clathrin-coat assembly would inhibit trafficking between which compartments?

4.A protein that is normally sent to the lysosome has all asparagine (single letter abbreviation N) residues replaced with lysine. What would happen to trafficking of this protein?

Answer 1

1. The retention of transmembrane proteins in the Endoplasmic membrane is carried out by short C-terminal sequences that contain two lysine residues (KKXX sequences). This sequence is specific motif present in the cytoplasmic region and interacts with COP1 protein complex that transports proteins between ER and golgi complex. If this sequence is removed from the protein it cannot interact with COP1 protein complex to be transported to golgi and thus the protein would be integrated into the endoplasmic membrane.

2. Most Endoplasmic reticulum(ER) resident proteins have a Lys-Asp-Glu-Leu (KDEL in one letter code) sequence at their C-terminus. Several experiments demonstrated that the KDEL sequence which acts as sorting signal, is both necessary and sufficient for retention in the ER. If this ER retention signal is removed from the protein, it is secreted from the cell. And if the signal is transferred to a protein that is normally secreted, the protein is now retained in the ER.

3. In the process of endocytosis where the eukaryotic cells internalize material from their surrounding environment, the internalization is achieved by the formation of membrane bound vesicles at the cell surface that arise by progressive invagination of the plasma membrane, followed by pinching off and release of free vesicles into the cytoplasm. This is done by processes of cellular eating (phagocytosis) and cellular drinking (pinocytosis).

In pinocytosis, selective and efficient uptake occurs when solutes are captured by specific high affinity receptors present on the cell surface that binds tightly to the extracellular macromolecule (the ligand) that it recognizes. This receptor ligand complexes then undergoes endocytosis, becoming a transport vesicle. Clathrin mediated endocytosis is one of the mechanism in pinocytosis process, known as receptor mediated endocytosis. In this mechanism macromolecules bind to cell surface receptors, accumulate in clathrin coated pits, ad enter the cell as a receptor and ligand complexes in clathrin coated vesicles. Each clathrin consits of three copies each of heavy chain and light chain, forming a three legged structure called a triskelion. Clathrin triskelions are the assembly units that assembles into coats on the cytoplasic side of the plasma membrae by interacting with its adaptor proteins.These clathrin coated vesicles from plasma membrane move to endosomes. They also transport proteins from trans golgi to late endosomes. Thus, blocking clathrin coat assembly would inhibit trafficiking between trans golgi and late endosomes(lysosomes) and also between plasma membrane and endosomes.

4.    Lysine is a common determinant for Mannose phosphorylation of lysosomal proteins, that enables the transport of soluble proteins between trans golgi to late endosomes by mannose 6-phosphate (M6P) signal sequence recognition.