No abstract

Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system.Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally imported into the organelle. The vast majority of these proteins possess the so-called peroxisomal targeting sequence 1 (PTS1), 1 a C-terminal tripeptide complying to the consensus sequence S/A/C-K/R/H-L/M (1-4). These PTS1-containing proteins are specifically targeted to the organelle matrix by Pex5p, the PTS1 receptor (5-9).According to current models of peroxisomal biogenesis, the Pex5p-mediated process of protein import can be divided into four steps. In the first step, newly synthesized PTS1-containing proteins interact with Pex5p in the cytosol. This protein-protein interaction involves the PTS1 signal on one side and the tetratricopeptide repeats domain of Pex5p on the other. The Pex5p-cargo protein complex is then recognized by the so-called docking machinery present in the peroxisomal membrane. Somewhere after this event, the PTS1-containing protein is released into the peroxisomal matrix. Finally, Pex5p is recycled back to the cytosol to catalyze additional rounds of transportation (reviewed in Refs. 10 -12).One obvious property of such cyclic mechanism is that it needs some form of energy input to function, and indeed, basically all the studies addressing this issue are unanimous in this respect: protein import into the peroxisomal matrix requires hydrolysis of ATP (13-21). However, the precise step(s) of this import pathway where energy input is necessary has not been firmly established. For instance, it is generally accepted that the step of protein translocation across the peroxisomal membrane requires ATP hydrolysis. Such conclusion derives from the fact that ATP depletion or the inclusion of non-hydrolysable ATP analogues in the several experimental systems used result in an inhibition of the peroxisomal import process. However, the possibility that recycling of Pex5p back to the cytosol is an ATP-dependent event and the rate-limiting step in all this process was never considered. In this scenario, inhibition of peroxisomal protein import by lack of ATP would resu...

Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early-onset muscle disease, caused by disease-associated variants in the laminin-α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently. The phenotype associated with LAMA2 defects has been expanded to include milder and atypical cases, being now collectively known as LAMA2-related muscular dystrophies (LAMA2-MD). Through an international multicenter collaborative effort, 61 new LAMA2 disease-associated variants were identified in 86 patients, representing the largest number of patients and new disease-causing variants in a single report. The collaborative variant collection was supported by the LOVD-powered LAMA2 gene variant database (https://www.LOVD.nl/LAMA2), updated as part of this work. As of December 2017, the database contains 486 unique LAMA2 variants (309 disease-associated), obtained from direct submissions and literature reports. Database content was systematically reviewed and further insights concerning LAMA2-MD are presented. We focus on the impact of missense changes, especially the c.2461A > C (p.Thr821Pro) variant and its association with late-onset LAMA2-MD. Finally, we report diagnostically challenging cases, highlighting the relevance of modern genetic analysis in the characterization of clinically heterogeneous muscle diseases.

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitro peroxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

According to current models of peroxisomal biogenesis, Pex5p cycles between the cytosol and the peroxisome transporting newly synthesized proteins to the organelle matrix. However, little is known regarding the mechanism of this pathway. Here, we show that Pex5p enters and exits the peroxisomal compartment in a process that requires ATP. Insertion of Pex5p into the peroxisomal membrane is blocked by anti-Pex14p IgGs. At the peroxisomal level, two Pex14p-associated populations of Pex5p could be resolved, stage 2 and stage 3 Pex5p, both exposing the majority of their masses into the organelle lumen. Stage 3 Pex5p can be easily detected only under ATP-limiting conditions; in the presence of ATP it leaves the peroxisomal compartment rapidly. Our data suggest that translocation of PTS1-containing proteins across the peroxisomal membrane occurs concomitantly with formation of the Pex5p-Pex14p membrane complex and that this is probably the site from which Pex5p leaves the peroxisomal compartment.