Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta‐oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium‐chain fatty acids are activated inside peroxisomes hby the acyl‐CoA synthetase Faa2p. On the other hand, long‐chain fatty acids are imported from the cytosolic pool of activated long‐chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.
The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Δ and pex19Δ, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexΔ mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.
The peroxisomal protein import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and -independent manner. Here we show that the two types of ubiquitination target different residues in the NH 2 -terminal region of Pex5p and we identify Pex4p (Ubc10p) as the ubiquitin-conjugating enzyme required for Ubc4p-independent ubiquitination. Whereas Ubc4p-dependent ubiquitination occurs on two lysine residues, Pex4p-dependent ubiquitination neither requires lysine residues nor the NH 2 -terminal ␣-NH 2 group. Instead, a conserved cysteine residue appears to be essential for both the Pex4p-dependent ubiquitination and the overall function of Pex5p. In addition, we show that this form of ubiquitinated Pex5p is susceptible to the reducing agent -mercaptoethanol, a compound that is unable to break ubiquitin-NH 2 group linkages. Together, our results strongly suggest that Pex4p-dependent ubiquitination of Pex5p occurs on a cysteine residue.
Most peroxisomal matrix proteins contain a carboxylterminal tripeptide that directs them to peroxisomes. Within limits, these amino acids may be varied, without loss of function. The specificity of this peroxisomal targeting signal (PTS1) is remarkable considering its small size and its relaxed consensus sequence. Moreover, several peroxisomal proteins have a PTS1-like signal that does not fit the reported consensus sequence. Because many of these PTS1 variants seem to be functional in a species-dependent or protein context-dependent manner, we investigated the PTS1 requirements in a homologous context, using Saccharomyces cerevisiae and endogenous peroxisomal malate dehydrogenase (MDH3). Peroxisomal import of the MDH3-PTS1 variants was tested qualitatively by the ability to complement the ⌬mdh3 mutant and quantitatively by subcellular fractionation. We observed efficient import of MDH3 into peroxisomes with a large variety of PTS1 tripeptides. Many of these variants do not fit the observed PTS1 requirements for heterologously expressed proteins, which suggests that additional domains in the protein may be of decisive importance whether or not a certain PTS1 variant is recognized by the components of the peroxisomal import machinery. Because we show that dimerization of MDH3 precedes import into the organelle, these domains are most likely conformational domains.
Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.
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