GRASPs Protein Mediated Golgi Membrane Stacking Metabolism And Structural Insight Into Endocytosis Protein SGIP1

Golgi apparatus is an intracellular organelle consists of flat membrane saccules and different size of vacuoles. The stacking of these cisternae ensures that membrane proteins and secretory proteins can be processed through the Golgi complex directionally and efficiently. The stacked architecture of the Golgi is stably maintained in animal cells, but becomes very dynamic during cell division. When cells enter mitosis, the Golgi stacks are disassembled into many small vesicles, allowing the partitioning of the Golgi components into the two daughter cells. After successful chromosome segregation, stacked cisternae are rebuilt to restore proper Golgi functions.Golgi assembly mainly depends on cis-GRASP55and medial-GRASP65protein, GRASP55can form complex with the Rab2effector golgin-45, while GM130and GRASP65interact and stable on the Golgi. Both GRASP65and GRASP55consist of an N-terminal GRASP domain composed of two tandem PDZ domains with high sequence homology, and a C-terminal Serine/Proline-rich (SPR) domain. Biochemical experiments have indicated that GRASP proteins forms oligomers, and the GRASP domain is sufficient for homotypic interactions.GRASP55and GRASP65is an important protein in the Golgi stacking has been widely studied and proved in recent years, however, the molecular mechanism involves in Golgi stacking is still unclear. In this paper, N terminal GRASP domain of GRASP55was crystalled and the structure was determined, the GRASP domain of GRASP55consists of two tandem PDZ domains, according to the initial sequence at N terminal, which is called PDZ1and PDZ2, each PDZ domain containing two alpha helix and five beta sheet. Compared with the crystal structure of GRASP55and GRASP65determined later by our lab, in both structures, the GRASP domain forms a dimer through homotypic interactions between the two PDZ2s; the tail of the GRASP domain, the immediate extension of the GRASP domain into the C-terminal region, also associates with PDZ1from the neighboring molecule. Experiments performed in vitro and in cells confirm that these two interfaces play important role in mediating oligomerization of the GRASPs and membrane tethering of Golgi. GRASP55and GRASP65protein by conformational changes in the two PDZ domain, effects on their own aggregation status, and further affect the stack of Golgi apparatus.Receptor mediated endocytosis plays a critical role in regulating cellular functions such as uptake of nutrients and down regulation of growth factor receptors. The process results from a receptor-ligand interaction. The ligands out of the cell first bound to specific plasma memberane receptors and then rapidly internalized by the cell. Internalisation follows clustering of the receptors in specialised regions of the cell surface called coated pits that invaginate to form intracellular coated vesicles.Recently a number of accessory proteins have been identified that participate in clathrin-mediated endocytosis such as the AP-2complex, EPS15, Fchol/2, espin, and SGDP1. SGIP1is initially discovered as a conserverd brain-enriched-protein regulating energy homeostasis and soon was identified as an endocytic adaptor. The N-terminal MP domain of SGIP1is responsible for plasma membrane binding, the middle PR domain interact with ITSN1and the C-terminal μHD domain recently declared forms complexes with the cytoplasmic domain of Calnexin. SGIP1plays important roles in function of clathrin-dependent endocytosis, yet no structural information has been reported, in this paper we report the structural studies of the C-terminal μHD domain of SGIP1. Well diffracting crystals were obtained by the sitting-drop vapour-diffusion method and X-ray diffraction data was collected to2.79A resolution using synchrotron radiation. SAD method was used to determine the structure and the structure provides us the basic molecular mechanism of how SGIP1was involved in the endocytosis process.