Coordination of membrane traffic in the early secretory pathway

Proteins and lipids are the major building blocks of cells, and as such are constantly produced within the cells or our body. The constant synthesis of these molecules is required for the growth and healthy maintenance of our tissues. Approximately one third of all proteins and practically all lipids are made in a cell compartment called the endoplasmic reticulum, from where most proteins and lipids are further transported to downstream compartments in specialized carriers called transport vesicles. This transport occurs along the secretory pathway, which delivers the proteins and many of the lipids to their final destination in the cell, or in some cases the molecules are released from the cell by secretion, as is the case for hormones, neurotransmitters, extracellular proteins that make up our skin and bone, and cholesterol-rich particles that circulate in our bloodstream. A major gatekeeping step in the secretory pathway is the exit of cargo proteins and lipids from the endoplasmic reticulum, a process mediated by specialized machinery called COPII, which acts together with accessory factors to ensure cargo is released in an efficient and properly controlled manner into carriers destined for the next compartment of the secretory pathway. This forward transport of cargo must also be balanced with transport in the reverse direction, which acts to recycle key factors and is mediated by a different machinery, called COPI. The mechanisms by which forward and backward transport are coordinated, which is vital for secretory pathway function, are unknown. Moreover, although we know defective secretory cargo transport negatively impacts upon cell viability and human health, the underlying mechanisms remain poorly defined.

This proposal focuses on a protein called Scyl1, whose mutation causes CALFAN syndrome in humans, a disorder that affects the nervous system, liver and skeleton. Presently we have only a poor understanding of Scyll1 function. We have obtained preliminary evidence that Scyl1 functions in conjunction with COPII, and that loss of Scyl1 disrupts cargo protein transport in the secretory pathway. These results point to a new role for Scyl1 in controlling exit of cargo proteins from the endoplasmic reticulum. Moreover, since Scyl1 is unique in its ability to bind to both COPII and COPI, it is a prime candidate for a factor coordinating forward and backward transport in the secretory pathway. Finally, because our preliminary results indicate that defective secretory cargo transport upon loss of Scyl1 causes induction of cell stress, investigating the underlying mechanisms of stress induction and its consequences will reveal how defective trafficking causes loss of cell viability, which is important for our understanding of human health. The work will not only be important for determining the mechanisms of CALFAN syndrome, but will have significance for our understanding of secretory cargo transport more generally, which is of fundamental importance to all cells.

The secretory pathway is fundamentally important, responsible for the synthesis, modification and transport of 30% of all cellular proteins. The entry portal to the secretory pathway is the endoplasmic reticulum (ER), and a key gatekeeping step in the pathway is the exit of cargo from this compartment, a process mediated by the COPII coat. The mechanisms of ER exit remain poorly understood, particularly in the case of bulky secretory cargoes such as LDL particles or procollagen. Moreover, it is not known how ER exit during anterograde secretory traffic is coupled with retrograde transport, mediated by COPI, which serves to recycle escaped ER residents and transport machinery. In preliminary studies we have found a novel interaction between Scyl1, whose mutation causes the rare disorder CALFAN syndrome, and the Sec31 subunit of the COPII coat, suggesting a role for Scyl1 in ER exit. Because Scyl1 also binds COPI, our results point to a role for Scyl1 in coupling COPII and COPI function in the early secretory pathway. We have found that depletion of Scyl1 causes defective transport of secretory cargo within the secretory pathway, and appears to trigger an ER stress response. Our findings lead us to propose that Scyl1 is a key trafficking protein, functionally coupling the COPII and COPI coats to ensure coordination of the early secretory pathway. We also hypothesize that disrupted secretory traffic, as seen upon Scyl1 loss, causes ER stress, that in turn negatively impacts upon cell viability. In this proposal we will test these hypotheses. We will identify the cargo proteins sensitive to loss of Scyl1, the mechanisms by Scyl1 coordinates secretory trafficking, and how loss of Scyl1 function results in ER and cellular stress signaling and the consequences of this upon cell viability. The results will increase our understanding of how the secretory pathway operates at the molecular level, and reveal how impaired trafficking can impact upon human health.

Wherever possible we will try to maximize the impact of our research. We will adopt several strategies to achieve this, as indicated below.

Public engagement
We envisage that findings of our current study will be of interest to the public at large given the importance of the processes we will investigate to human health. We will communicate our findings to the public via the open access University website and through the Faculty of Biology, Medicine and Health (FBMH) research brochure. We will also notify the dedicated Faculty press officers of our findings at around the point of publication in academic journals. The press officer will then contact local and national news agencies, and prepare press releases that these agencies can use. We will also contact appropriate stakeholders including CALFAN patients and their families and associated health care professionals. The Lowe lab has participated in several public engagement initiatives and we plan for this to continue. The lab has hosted several A-level students at different times as part of the Nuffield bursary scheme. The FBMH continues to participate in the Nuffield scheme and we envisage hosting another 1-2 A-level students should the current application be successful. Members of the Lowe lab have participated in several public engagement exercises involving visiting schoolchildren, hosted at the Manchester Museum with whom the University has close links. The High lab has already written specialist material for the Young Persons University of Manchester portal and we will build on this expertise to create accessible bespoke content about our work on Scyl1.

Exploitation and application
The work outlined in the proposal is basic research. Thus it is not trivial to realize the short and long term benefit of the work in terms of direct commercial or clinical exploitation. However, the scientific data obtained will increase the knowledge of UK and international scientists in the academic, clinical and commercial sectors. This contribution to knowledge will lead to improved understanding at the level of basic understanding of cellular function, and will also contribute towards an improved understanding of mechanisms of disease and ageing. This increase in knowledge will aid the design of better diagnosis and treatments for patients. This is particularly relevant to CALFAN syndrome, but may also be applicable to other neurodegenerative or liver diseases. With regard to the latter, because CALFAN syndrome causes liver cirrhosis and liver failure, both of which are observed in alcohol and drug-induced liver disease, our findings may also have applications in the diagnosis or treatment of these common societal diseases. Finally, because most biologics are synthesized in the secretory pathway, our findings may be exploited by the bio-pharmaceutical industry.

Training
The technical and intellectual knowledge acquired by the post-doctoral researcher during the course of the proposed work will equip this person to pursue a future career as an independent scientist. The researcher will also learn transferable skills that will increase his employability. Such highly trained individuals will contribute to the UK knowledge and skill base and ultimately the UK economy.