Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein-a mutated bacterial dehalogenase which is fused to the protein of interest-and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of preexisting peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.
Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.Since the discovery of the ubiquitin-conjugating cascade nearly 30 years ago, thousands of proteins have been shown to be modified by ubiquitin (1, 2). In many cases ubiquitination of a protein is linked to its proteasomal degradation (3), whereas in a growing number of examples, ubiquitination of a protein is used as a transient modification to modulate its biological properties (for a review see Ref. 4). Regardless of the final outcome, it is generally assumed and in many cases demonstrated that ubiquitin is covalently attached through an amide bond involving the carboxyl group of the last glycine of ubiquitin on one hand, and an amino group of the targeted protein on the other (5). Recent findings from several laboratories, however, suggest that this rule is not always valid, and proteins ubiquitinated at serines and threonines (yielding oxyesters) or even cysteines (forming thiol esters) have been identified (6 -10).Protein ubiquitination at cysteine residues is a particularly puzzling phenomenon for two reasons. First, on a thermodynamic basis it is the least favorable event (the approximate free energy changes for acyl shifts from a thiol ester to a thiol, alcohol, and amine are 0, Ϫ2.4, and Ϫ11 kcal/mol, respectively (11, 12)). Second, although data on the half-lives of ubiquitin-protein thiol ester conjugates under physiologically relevant conditions are scarce, it is known that ubiquitin thiol esters are easily disrupted by nucleophiles such as GSH (13), raising the possibility that, to some degree, proteins subjected to this kind of conjugation may undergo futile ubiquitination/deubiquitination cycles. Thus, a thiol ester bond appears not to be the most efficient way to link ubiquitin to a protein, unless, of course, the aim is to create an activated (easily transferable) form of ubiquitin, as is in fact the case with ubiquitin-activating enzymes (E1s), 4 ubiquitin-conjugating enzymes (E2s), and some ubiquitin ligases (E3s) (2).In the last years we have been characterizing Pex5p, one of the three presently known proteins claimed to be ub...
Three new therapies for spinal muscular atrophy (SMA) have been approved by the United States Food and Drug Administration and the European Medicines Agency since 2016. Although these new therapies improve the quality of life of patients who are symptomatic at first treatment, administration before the onset of symptoms is significantly more effective. As a consequence, newborn screening programs have been initiated in several countries. In 2018, we launched a 3-year pilot program to screen newborns for SMA in the Belgian region of Liège. This program was rapidly expanding to all of Southern Belgium, a region of approximately 55,000 births annually. During the pilot program, 136,339 neonates were tested for deletion of exon 7 of SMN1, the most common cause of SMA. Nine SMA cases with homozygous deletion were identified through this screen. Another patient was identified after presenting with symptoms and was shown to be heterozygous for the SMN1 exon 7 deletion and a point mutation on the opposite allele. These ten patients were treated. The pilot program has now successfully transitioned into the official neonatal screening program in Southern Belgium. The lessons learned during implementation of this pilot program are reported.
This report represents the second PEX14-deficiency associated with Zellweger syndrome and the first documentation of a PEX14-deficient patient with detailed clinical follow-up and biochemical, morphological, and radiological data.
BackgroundPeroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes.ResultsHere we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology.ConclusionThese observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways.
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