The peroxisomal protein import machinery displays a preference for monomeric substrates

Abstract

Peroxisomal 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 models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

The first minutes in the life of a peroxisomal matrix protein

“…This behavior suggests, on one hand, that these monomers are already at least partially folded and, on the other, that folding of monomers and their oligomerization to yield the native enzymes are not physically coupled events. The same conclusion is probably valid for monomer folding and monomer-PEX5 interaction because some active oligomeric peroxisomal proteins can be detected in the cytosol of cells lacking PEX5 [64][65][66] and the in vitro protein synthesis system used in the experiments referred to above has essentially no endogenous PEX5 [34,62,63]. Thus, it appears that newly synthesized proteins are released by the cytosolic chaperone machinery as soluble monomeric proteins independently of PEX5.…”

“…In vitro import assays aiming at comparing the import efficiencies of monomeric and oligomeric versions of three different proteins also pointed into the same direction: the monomeric versions of Cucurbita pepo isocitrate lyase, mouse ACOX1 and urate oxidase (UOX) were found to be better import substrates than the corresponding oligomeric forms [56,63]. Actually, for ACOX1 and UOX no evidence for in vitro import of their oligomeric forms could be obtained [63].…”

“…), all these studies used experimental conditions that lead to very high levels of the characterized proteins. As discussed recently, these conditions can potentially lead to the titration of the peroxisomal protein import machinery (PIM), and thus to the ectopic (i.e., cytosolic) oligomerization of the reporter proteins [63]. Second, in only one case was the import kinetics of the reporter oligomeric protein documented [43].…”

“…An interesting property of several peroxisomal homo-oligomeric proteins regards the fact that they can be detected as soluble monomeric proteins immediately after synthesis, both in vivo [59][60][61] and in vitro [34,62,63]. This behavior suggests, on one hand, that these monomers are already at least partially folded and, on the other, that folding of monomers and their oligomerization to yield the native enzymes are not physically coupled events.…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…However, the import machinery prefers monomeric proteins (Freitas et al, 2015), and PEX5 binding to catalase (Freitas et al, 2011), acyl-CoA oxidase1, and urate oxidase (Freitas et al, 2015) prevents oligomerization of these cargo proteins.…”