Investigation of Rabs and cargo receptors as key regulators of vesicle traffic

Eukaryotic cells are subdivided into organelles with unique physicochemical compositions and functions. Establishment and maintenance of these organelles requires selective exchange of material by vesicle trafficking. Transport vesicles are formed by cytoplasmic coat protein complexes which shape the membrane, and play a further role in the selection of transmembrane cargo proteins. Transmembrane cargo receptors select cargo in the luminal compartment and bind to these coat proteins through signals in their cytoplasmic domains. Once formed, vesicles recognise and fuse with a target organelle to deliver their content. This final process is tightly regulated by small GTPases of the Rab superfamily. Here, I will describe my work investigating the role of specific Rab GTPase pathways and the KDEL cargo receptor in trafficking in the secretory and endocytic pathways in mammalian cells. There are over 60 Rabs in human cells, each of which is activated by a specific GDP-GTP exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP) at a defined membrane location. For many of these Rabs the GEF and GAP were unknown at the outset of this work in which I have identified and characterised two structurally related families of Rab GEFs, the DENN and tri-longin domain proteins. The DENN family, expressed only in metazoans, which has 17 members acting on 10 different Rabs. The tri-longin family, comprises 3 heterodimeric GEF complexes: Mon1-Ccz1 for the late endosomal, lysosomal Rab7, Hps1-Hps4 (BLOC-3) which activates Rab32/Rab38 in melanogenesis and Intu-Fuz, part of CPLANE complex, which activates Rab23 at cilia. Additionally, I have shown that the unrelated Rab3GAP complex is a GEF for the ancestral Rab18 regulating the structure of the endoplasmic reticulum. In the final part of this work, I have explored how the integrity of the early secretory system depends on selective export and retrieval of proteins between the ER and the Golgi. An essential component of this system is the transmembrane cargo receptor for KDEL retrieval signals. Here I describe the structure of the KDEL receptor and mechanism by which KDEL cargo binding triggers a conformational change exposing a signal for the COP I vesicle coat