Functional analysis of a family of TANGO1-like proteins. What is the influence on transport of large cargos and are so-called megacarriers generated?

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Higher eukaryotes produce a wide range of proteins and molecules, a large portion of these have their destination outside of the cell. In order to reach the outside, these proteins are translated into the endoplasmatic reticulum, are transported to the Golgi and from there to the cell surface. The transport from the endoplasmatic reticulum to the Golgi is facilitated by COPII vesicles, that have a defined size of about 60-90nm. Interestingly, some of the transported proteins are quit large. An example is Collagen VII that comes in the form of a 400nm long, rigid triple helix. It has been speculated that these large cargos are transported in megacarriers, larger than usual vesicles. So far it is neither known whether these vesicles truly exist, nor how they form.It is known however, that the ER exit site localized protein TANGO1 plays a role in the transport of Collagen VII. When TANGO1 is depleted, cells can no longer shuttle Collagen VII from the endoplasmatic reticulum to the Golgi. Interestingly, bulk protein secretion is not affected in these cells. Trough sequence comparison I discovered a family of TANGO1-like (Tali) proteins that like TANGO1 localize to ER exit sites. I want to explore if these proteins, together with TANGO1, play a role in the generation of megacarriers or whether they are needed in other steps of vesicle biogenesis at the endoplasmatic reticulum.An additional aim of the proposal is to investigate how large proteins are transported anyway. To clarify the involvement of megacarriers in the transport of large cargo and to gain insight into the mechanism, I will in Vitro reconstitute vesicle biogenesis at the level of the ER.