**6. Summary**

Despite the similarity in requirements for membrane dynamics in the processes of exocytosis and autophagy, correlations between the molecular machinery used for both of these processes are only beginning to be elucidated. The exocytic and autophagic functions of cells are critical for the maintenance of cell homeostasis and the exchange of membrane between intracellular compartments and the cell surface. In addition, the fusion and fission events that remodel the exocytic vesicle and the autophagosome are likely to require much of the same molecular machinery. Therefore, it is likely that there is co-ordinated control of these two processes to ensure that they can be regulated with respect to each other. Members of the exocyst complex, and some autophagy related proteins, have already been shown to have functions in their opposite processes, and the involvement of the Ral small GTPases in the global control of exocytosis and autophagy mirrors the role of Rab small GTPases in the control of endosome trafficking. There are many intriguing questions brought about by recent findings. What is the decision making signal that diverges the components of the shared machinery from one pathway to another? Is there a common upstream signal for both pathways, be it through the insulin receptor/mTOR (Webber & Tooze, 2010), MAPK (Webber & Tooze, 2010), redox (Lee *et al*., 2012), or are there combinations of these signals? Or is there a yet to be defined intrinsic factor of the autophagic or exocytic membrane, with a changing affinity for vesicular compartments? This is a very interesting time to be exploring the intersection of the exocytosis and autophagy pathways, particularly while we are is still looking for the key controllers of cellular homeostasis in cancers, neurodegenerative and immune disorders.

## **7. References**


lysosome-regulated exocytosis is not restricted to osteoclasts involved in bone remodelling, and has been described for lysosome related organelles in many other specialist cell types, such as melanosomes in melanocytes and lytic granules in neutrophils (Blott & Griffiths, 2002; Chen *et al*., 2012; Luzio *et al*., 2007). In yeast grown under conditions of nitrogen starvation, autophagy genes are required for the secretion of an acyl coenzyme A binding protein (Acb1) (Bruns *et al*., 2011; Duran *et al*., 2010; Manjithaya *et al*., 2010; Skinner, 2010). This unconventional route of secretion is initiated at sites that are positive for the Golgi assembly and stacking protein (GRASP65) homologue 1 (Grh1), which attracts core autophagy-related proteins Atg9 and Atg8 to a novel compartment (Bruns *et al*., 2011). These Acb1-containing autophagosomes then evade fusion with the lytic vacuole, fusing instead with recycling endosomes to form multivesicular body carriers, which then fuse with the plasma membrane in a t-SNARE Sso1 dependent fashion, to release Acb1. It is still not clear how beneficial or economical it

Despite the similarity in requirements for membrane dynamics in the processes of exocytosis and autophagy, correlations between the molecular machinery used for both of these processes are only beginning to be elucidated. The exocytic and autophagic functions of cells are critical for the maintenance of cell homeostasis and the exchange of membrane between intracellular compartments and the cell surface. In addition, the fusion and fission events that remodel the exocytic vesicle and the autophagosome are likely to require much of the same molecular machinery. Therefore, it is likely that there is co-ordinated control of these two processes to ensure that they can be regulated with respect to each other. Members of the exocyst complex, and some autophagy related proteins, have already been shown to have functions in their opposite processes, and the involvement of the Ral small GTPases in the global control of exocytosis and autophagy mirrors the role of Rab small GTPases in the control of endosome trafficking. There are many intriguing questions brought about by recent findings. What is the decision making signal that diverges the components of the shared machinery from one pathway to another? Is there a common upstream signal for both pathways, be it through the insulin receptor/mTOR (Webber & Tooze, 2010), MAPK (Webber & Tooze, 2010), redox (Lee *et al*., 2012), or are there combinations of these signals? Or is there a yet to be defined intrinsic factor of the autophagic or exocytic membrane, with a changing affinity for vesicular compartments? This is a very interesting time to be exploring the intersection of the exocytosis and autophagy pathways, particularly while we are is still looking for the key controllers of

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**6** 

Cees M.J. Sagt

*The Netherlands* 

*DSM Biotechnology Center, Delft* 

**Peroxicretion, a Novel Tool for** 

**Engineering Membrane Trafficking** 

The production of proteins by recombinant micro-organisms has been possible since the late 70's. Since then enormous steps have been made to improve protein titers and to expand the range of possible proteins to be produced. The development of genetic modification tools and improved ease of use of cloning strategies have played an important role in this. The time to construct a strain and develop a cost-effective bioprocess has decreased significantly. Concurrently, sequencing data, which serve as a library for donor genes to be overexpressed, have increased exponentially. Therefore, the speed and flexibility to engineer custom-made protein factories has improved tremendously during the last decades. As in any developed technology, a certain degree of standardization is desired. In order to make this ambition more tangible the concept of cell factories is often used. This concept is based on the principle that the cell factory should be able to produce any protein to a certain desired level. Based on the concept of standardized expression vectors and expression hosts the basics of this cell factory concept were built. However, in reality this concept proved to be far too simple, as a standardized input does not result in a standardized output. This is caused by the immense complexity and dynamics of cellular systems. Even when all components are described, by sequencing the genome, the interaction, compartmentalization and dynamics of these components are largely unknown. A striking example is the difference between homologous protein expression which are produced to levels up to 50 g/l in filamentous fungi whereas heterologous proteins are produced at levels which are usually 100-1000 fold less 1. So even when identical tools are used in a

standardized approach the final result can vary over several orders of magnitude.

The general consensus is that this large difference is linked to cellular events and responses which are caused by the overexpression of the heterologous proteins 2, therefore the scientific community, together with biotech industries, have been studying these effects for several decades. The cellular stress responses and intracellular events which are linked to the use of cells as protein production factories are very well described 3-9. This profound insight in the molecular mechanism of the cellular stress reactions has facilitated the increase in expression and secretion levels of some heterologous proteins. However, levels comparable to homologous proteins have not been reached 1, 10. It is evident that, when even more demanding sources of biodiversity are tapped, like intracellular proteins, the engineering of a robust and versatile cell factory which is able to produce this wide range of

**1. Introduction** 

proteins becomes a major challenge.

