טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAbutbul Ionita Inbal
SubjectStructure-Function Studies of Proteins of the Dynamin Family
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Dganit Danino
Full Thesis textFull thesis text - English Version


Abstract

Proteins of the dynamin family are large GTPases found in animal, yeast and plant cells. They share high sequence homology, structural motifs, biochemical characteristics, and the ability to self-assemble into ordered structures and interact with cellular membranes. At the same time, dynamin family members are implicated in diverse fundamental cellular processes. Here we present structure-function studies of distinct highly-ordered complexes, which are customized to fit diverse cellular functions. The study focuses on three family members: dynamin, MxA, and Mgm1.


Dynamin is considered to be the prototype for proteins of the dynamin family. It is essential for many biological functions that require fission, such as endocytosis, synaptic recycling, and vesicle trafficking. Dynamin assembles into helical structures at the necks of budding vesicles, and assists the release of vesicles from the membrane upon GTP binding and hydrolysis. In-vitro dynamin wraps around liposomes and transform them into long helical tubes, with a diameter similar to that of endocytic buds. Upon GTP hydrolysis, dynamin applies force on the underlying membrane and constricts, decreasing the distance between bilayers, and facilitating fission.


MxA is an antiviral protein that inhibits human and other viral pathogens. Despite intense research, MxA mechanism of antiviral activity is still unclear. It is suggested that MxA is antivirally-active as a monomer, and GTPase activity may be required for monomer release from assembled MxA. However, it is not clear whether GTP binding or hydrolysis are needed for the release of MxA monomers from MxA assemblies. We examined the protein assemblies at different stages along the GTP cycle, to determine the conditions controlling the self-assembly. Our results indicate that GTP hydrolysis and not binding is the stage in which MxA disassembles into monomers.


Mgm1 function in mitochondrial membrane fusion and the formation and maintenance of cristae structures. The protein mechanism of action in these processes is yet to be discovered. s-Mgm1 was successfully expressed and purified only recently and to date very little work examined the protein characteristics in-vitro. Preliminary work in our lab focused on s-Mgm1-lipid interactions. We found that s-Mgm1 forms unique membrane associated structures that are novel for the dynamin family. s-Mgm1 does not tubulate single liposomes like all known dynamins. Instead, it crystallizes onto the leaflets of liposomes, then bridges the membranes of neighboring liposomes and anchors them at a fixed distance. s-Mgm1 structures of bridged liposomes strikingly resemble mitochondrial cristae structures and could be the first step towards fusion.