Question

Insulin is a hormone made and secreted by the beta cells in the pancreas. You have generated various beta-cell mutant cell lines as described below. Explain whether the mutation will have an effect on the insulin secretion. If so, what step(s) of the secretory pathway is likely to be affected by the mutation? Explain your answers. Cell line A: A small GTPase Arf1 (functions on Golgi membrane) is mutated that the GTPase domain of the protein is inactive Cell line B: Cells do not express Arf-GEF Cell line C: Cells do not express t-SNARE on the plasma membrane

Answer 1

C. Proinsulin after its synthesis in the ER, will from the ER and will be sorted in the Trans GOLGI network (TGN) into immature secretory granules. Proinsulin is packaged in TGN into vesicles after it is converted to hexamer. Proinsulin is converted to insulin into mature insulin and C-peptide in mature vesicles. The mature secretory vesicles will then move to plasma membrane, fuse and release insulin at the plasma membrane for incorporation.

Proinsulin packaging into clathrin coated compartment during the exit in TGN, is regulated by AP-1 complex and Arf-1. ARf-1 is ribosylation factor which binds GTP. It binds to GTP and GTP can then be hydrolyzed to GDP. When ARf-1 binds to GTP, it is activated. It can then interact with effectors. However, when GTP is hydrolyzed to GDP, it has lesser affinity for effector molecules and will result in dissociation. Arf-1 binds to Arfaptin-1. This Arf1 bound Arfaptin 1 is recruited to Golgi membrane in GTP bound form. It is then phosphorylated by protein kinase D. These molecules are involved in fission of the secretory granules in TGN.

If the GTPase domain of Arf-1 is mutated, then Arf1 will be always active as GTP will not be cleaved to GDP. Hence, Arf1 will bind to Arfaptin and there is the processing in TGN. Thus, the TGN process is always active due to Arf1 being in FTP bound state, leading to increased insulin secretion. TGN transit of proinsulin is affected.

B. ARF-GEFs are guanine nucleotide exchange factors can associate with plasma/Golgi membrane under specific conditions. They have an N-terminal coiled-coil domain, a central Sec7 like domain, and a C-terminal pleckstrin homology domain. The Arf-1-GEFs are involved in exchange of GDP for GTP. If cells do not express Arf1-GEF, then GDP cannot be exchanged for GTP. This will result in no trans-Golgi transport as Arf1 will be inactive due to it being in GDP bound state. Thus, insulin secretion will decrease. The mutation affects Trans Golgi network transit.

C. SNARE proteins are involved in fusion of insulin containing secretory vesicles with the plasma membrane. The tSNAREs are target snares present on plasma membrane. They mediate fusion between the plasma membrane and the vesicle membrane via interaction with vSNARE. If tSNARE is not expressed on the plasma membrane, there will be no fusion of the secretory vesicles with plasma membrane. Thus, secretion of insulin will be inhibited. This mutation will affect the exocytosis part of the pathways, which is fusion of mature secretory vesicle with plasma membrane.