Peroxicretion: a novel secretion pathway in the eukaryotic cell

Cees Mj Sagt,Jan Metske Van Der Laan,Miranda P Hartog,Michiel Akeroyd,Ingeborg M Minneboo,Robbert A Damveld,Francisca A Luesken,Ida Van Der Klei,Peter J Ten Haaft,Marten Veenhuis,Johannes H De Winde,Thibaut J Wenzel

doi:10.1186/1472-6750-9-48

Abstract

BackgroundEnzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.ResultsPeroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.ConclusionOur results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

Highlights

Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing
To confirm that PTS1 signals will result in peroxisomal localization in A. niger we have identified a Pex5 ortholog in the genome of A. niger (Genbank 4989140)
Amino acids important for PTS1 recognition are conserved in the Pex5 ortholog, suggesting that the presence of an -SKL sequence at the C-terminus of model proteins will lead to peroxisomal localization

Summary

Introduction

Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. The specificity of intracellular membrane trafficking is determined by multiple layers of control mechanisms that ensure that only appropriate organelles fuse with specific target compartments. These include Rab-GTPases [1] operating in conjunction with polyphosphoinositides [2] and Rab effectors [3] that frequently include multiprotein complexes. The ER supplies the secretory route with membrane enclosed vesicles which travel from the ER via the Golgi towards the cell membrane

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Conclusion