Peroxisomal proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the peroxisome by cycling receptors. There are two main receptors: PEX19 is the receptor/chaperone for intrinsic membrane proteins; PEX5, alone or with the help of the adaptor protein PEX7, is the receptor/translocator for proteins destined to the matrix of the organelle. Our main aim has been to understand the mechanisms of these two protein sorting pathways. Using in vitro import systems developed in our laboratory, we have shown that PEX5 becomes transiently inserted into the peroxisomal membrane docking/translocation machinery during the transport cycle. Our data further suggest that protein-protein interactions involving PEX5 on one side, and the membrane machinery on the other, provide the energy for the cargo translocation process. Following this step, PEX5 is extracted from the peroxisomal membrane machinery in an ATP-requiring step. Remarkably, this requires monoubiquitination of PEX5 at a conserved cysteine residue. Finally, the ubiquitin moiety is removed from the cytosolic PEX5 conjugate probably by a combination of enzymatic and non-enzymatic mechanisms. Our goal at present is to understand why this unconventional ubiquitination is used in this pathway. Future research goals: We will continue to study the mechanism of protein sorting into peroxisomes. Efforts will be focused on PEX7, an adaptor protein that increases the range of cargo proteins recognized and transported by PEX5. In parallel, we will try to understand how PEX5 interacts directly with most cargo proteins and, after insertion into the peroxisomal docking/translocation machinery, releases them into the organelle matrix in a process that apparently does not require ATP-hydrolysis. Besides the work on peroxisomal biogenesis we are also developing a new research line on protein regulation by ubiquitin and ubiquitin-like molecules. Selected references: Rodrigues TA, Alencastre IS, Francisco T, Brites P, Fransen M, Grou CP, Azevedo JE.(2014) A PEX7-centered perspective on the peroxisomal targeting signal type 2-mediated protein import pathway. Mol. Cell. Biol. 34(15):2917-28. (doi: 10.1128/MCB.01727-13) Francisco T, Rodrigues TA, Pinto MP, Carvalho AF, Azevedo JE, Grou CP. (2014) Ubiquitin in the peroxisomal protein import pathway.Biochimie 98:29-35. (doi: 10.1016/j.biochi.2013.08.003) Francisco T, Rodrigues TA, Freitas MO, Grou CP, Carvalho AF, Sá-Miranda C, Pinto MP, Azevedo JE. (2013) A cargo-centered perspective on the PEX5 receptor-mediated peroxisomal protein import pathway.J. Biol. Chem. 288(40):29151-9. (doi: 10.1074/jbc.M113.487140) Pinto MP, Carvalho AF, Grou CP, Rodríguez-Borges JE, Sá-Miranda C, Azevedo JE. (2012) Heat shock induces a massive but differential inactivation of SUMO-specific proteases. Biochim. Biophys. Acta 1823(10):1958-66. (doi: 10.1016/j.bbamcr.2012.07.010). Grou CP, Francisco T, Rodrigues TA, Freitas MO, Pinto MP, Carvalho AF, Domingues P, Wood SA, Rodríguez-Borges JE, Sá-Miranda C, Fransen M, Azevedo JE. (2012) Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate. J. Biol. Chem. 287(16):12815-27. (doi: 10.1074/jbc.M112.340158) Freitas MO, Francisco T, Rodrigues TA, Alencastre IS, Pinto MP, Grou CP, Carvalho AF, Fransen M, Sá-Miranda C, Azevedo JE. (2011) PEX5 protein binds monomeric catalase blocking its tetramerization and releases it upon binding the N-terminal domain of PEX14. J. Biol. Chem. 286(47):40509-19. (doi: 10.1074/jbc.M111.287201)

Organelle Biogenesis and Function