12 documents found, page 1 of 2
A mechanistic perspective on pex1 and pex6, two aaa+ proteins of the peroxisoma...
Pedrosa, AG; Francisco, T; Ferreira, MJ; Rodrigues, TA; Barros-Barbosa, A; Azevedo, JEIn contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—...
Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6...
Pedrosa, AG; Francisco, T; Bicho, D; Dias, AF; Barros-Barbosa, A; Hagmann, V; Dodt, G; Rodrigues, TA; Azevedo, JEPEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation c...
Determining the topology of peroxisomal proteins using protease protection assays
Francisco, T; Dias, AF; Pedrosa, AG; Grou, CP; Rodrigues, TA; Azevedo, JEProtease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be c...
Protein transport into peroxisomes: Knowns and unknowns
Francisco, T; Rodrigues, TA; Dias, AF; Barros-Barbosa, A; Bicho, D; Azevedo, JEPeroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the “plunger” - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the “barrel” - into...
The peroxisomal matrix protein translocon is a large cavity-forming protein ass...
Dias, AF; Rodrigues, TA; Pedrosa, AG; Barros-Barbosa, A; Francisco, T; Azevedo, JEA remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5– cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur rema...
A cell-free organelle-based in vitro system for studying the peroxisomal protei...
Rodrigues, TA; Francisco, T; Dias, AF; Pedrosa, AG; Grou, CP; Azevedo, JEHere we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized 35 S-labeled reporter protein...
The first minutes in the life of a peroxisomal matrix protein
Dias, AF; Francisco, T; Rodrigues, TA; Grou, CP; Azevedo, JEIn the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric ...
Revisiting the intraperoxisomal pathway of mammalian PEX7
Rodrigues, TA; Grou, CP; Azevedo, JENewly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM duri...
The peroxisomal protein import machinery displays a preference for monomeric su...
Freitas, M; Francisco, T; Rodrigues, TA; Lismont, C; Domingues, P; Pinto, MP; Grou, CP; Fransen, M; Azevedo, JEPeroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current ...
Factors involved in ubiquitination and deubiquitination of PEX5, the peroxisoma...
Rodrigues, TA; Francisco, T; Carvalho, A; Pinto, MP; Grou, CP; Azevedo, JEPeroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by the soluble factor PEX5. Besides a role as a receptor, and probably as a chaperone, PEX5 also holds the key to the matrix of the organelle. Indeed, the available data suggest that PEX5 itself pushes these proteins across the peroxisomal membrane using as driving force the strong protein-prote...