Multiple Pathways for Protein Transport to Peroxisomes

Peroxisomes are unique among the organelles of the endomembrane system. Unlike other organelles that derive most if not all of their proteins from the ER (endoplasmic reticulum), peroxisomes contain dedicated machineries for import of matrix proteins and insertion of membrane proteins. However, peroxisomes are also able to import a subset of their membrane proteins from the ER. One aspect of peroxisome biology that has remained ill defined is the role the various import pathways play in peroxisome maintenance. In this review, we discuss the available data on matrix and membrane protein import into peroxisomes

Yeast peroxisomes multiply by growth and division

Peroxisomes can arise de novo from the endoplasmic reticulum (ER) via a maturation process. Peroxisomes can also multiply by fission. We have investigated how these modes of multiplication contribute to peroxisome numbers in Saccharomyces cerevisiae and the role of the dynamin-related proteins (Drps) in these processes. We have developed pulse-chase and mating assays to follow the fate of existing peroxisomes, de novo–formed peroxisomes, and ER-derived preperoxisomal structures. We find that in wild-type (WT) cells, peroxisomes multiply by fission and do not form de novo. A marker for the maturation pathway, Pex3-GFP, is delivered from the ER to existing peroxisomes. Strikingly, cells lacking peroxisomes as a result of a segregation defect do form peroxisomes de novo. This process is slower than peroxisome multiplication in WT cells and is Drp independent. In contrast, peroxisome fission is Drp dependent. Our results show that peroxisomes multiply by growth and division under our assay conditions. We conclude that the ER to peroxisome pathway functions to supply existing peroxisomes with essential membrane constituents

A dual function for Pex3p in peroxisome formation and inheritance

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum

NBP (nitrogen-containing bisphosphonate) drugs protect against excessive osteoclast-mediated bone resorption. After binding to bone mineral, they are taken up selectively by the osteoclasts and inhibit the essential enzyme FDPS (farnesyl diphosphate synthase). NBPs inhibit also growth of amoebae of Dictyostelium discoideum in which their target is again FDPS. A fusion protein between FDPS and GFP (green fluorescent protein) was found, in D. discoideum, to localize to peroxisomes and to confer resistance to the NBP alendronate. GFP was also directed to peroxisomes by a fragment of FDPS comprising amino acids 1–22. This contains a sequence of nine amino acids that closely resembles the nonapeptide PTS2 (peroxisomal targeting signal type 2): there is only a single amino acid mismatch between the two sequences. Mutation analysis confirmed that the atypical PTS2 directs FDPS into peroxisomes. Furthermore, expression of the D. discoideum FDPS–GFP fusion protein in strains of Saccharomyces cerevisiae defective in peroxisomal protein import demonstrated that import of FDPS into peroxisomes was blocked in a strain lacking the PTS2-dependent import pathway. The peroxisomal location of FDPS in D. discoideum indicates that NBPs have to cross the peroxisomal membrane before they can bind to their target

Repulsive long-range forces between anisotropic atoms and dielectrics

We investigate long-range forces between atoms with anisotropic electric polarizability interacting with dielectrics having anisotropic permittivity in the weak-coupling approximation. Unstable configurations in which the force between the objects is repulsive are constructed. Such configurations exist for three anisotropic atoms as well as for an anisotropic atom above a dielectric plate with a hole whose permittivity is anisotropic. Apart from the absolute magnitude of the force, the dependence on the configuration is qualitatively the same as for metallic objects for which the anisotropy is a purely geometric effect. In the weak limit closed analytic expressions for rather complicated configurations are obtained. The non-monotonic dependence of the interaction energy on separation is related to the fact that the electromagnetic Green's dyadic is not positive definite. The analysis in the weak limit is found to also semi-quantitatively explain the dependence of Casimir forces on the orientation of anisotropic dielectrics observed experimentally. Contrary to the scalar case, irreducible electromagnetic three-body energies can change sign. We trace this to the fact that the electromagnetic Green's dyadic is not positive definite.Comment: 9 page

Conductance Fluctuations of Generic Billiards: Fractal or Isolated?

We study the signatures of a classical mixed phase space for open quantum systems. We find the scaling of the break time up to which quantum mechanics mimics the classical staying probability and derive the distribution of resonance widths. Based on these results we explain why for mixed systems two types of conductance fluctuat ions were found: quantum mechanics divides the hierarchically structured chaotic component of phase space into two parts - one yields fractal conductance fluctuations while the other causes isolated resonances. In general, both types appear together, but on different energy scales.Comment: restructured and new figure

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Δ mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Δ cells suggest the existence of various levels of control involved in the partitioning of peroxisomes

Laser-induced splittings in the nuclear magnetic resonance spectra of the rare gases

Circularly polarized laser field causes a shift in the nuclear magnetic resonance (NMR) spectra of all substances. The shift is proportional to the intensity of the laser beam and yields oppositely signed values for left- and right-circularly polarized light, CPL -/+, respectively. Rapid switching -- in the NMR time scale -- between CPL+ and CPL- gives rise to a splitting of the NMR resonance lines. We present uncorrelated and correlated quadratic response calculations of the splitting per unit of beam intensity in the NMR spectra of $^{21}$Ne, $^{83}$Kr, and $^{129}$Xe. We study both the regions far away from and near to optical resonance and predict off-resonance shifts of the order 0.01, 0.1, and $1\times 10^{-6}$ Hz for $^{21}$Ne, $^{83}$Kr, and $^{129}$Xe, respectively, for a beam intensity of 10 W/cm$^2$. Enhancement by several orders of magnitude is predicted as the beam frequency approaches resonance. Only then can the effect on guest $^{129}$Xe atoms be potentially useful as a probe of the properties of the host material.Comment: 5 pages, 1 figur

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae.

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells. Degradation depended on Atg11 and the pexophagy receptor Atg36, which mediates degradation of superfluous peroxisomes. Mutants of PEX1, PEX6, and PEX15 accumulate ubiquitinated receptors at the peroxisomal membrane. This accumulation has been suggested to trigger pexophagy in mammalian cells. We show by genetic analysis that preventing this accumulation does not abolish pexophagy in Saccharomyces cerevisiae. We find Atg36 is modified in pex1Δ cells even when Atg11 binding is prevented, suggesting Atg36 modification is an early event in the degradation of dysfunctional peroxisomal structures in pex1Δ cells via pexophagy

Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane. We name this protein Atg36 as its absence blocks pexophagy, and its overexpression induces pexophagy. We have isolated pex3 alleles blocked specifically in pexophagy that cannot recruit Atg36 to peroxisomes. Atg36 is recruited to mitochondria if Pex3 is redirected there, where it restores mitophagy in cells lacking the mitophagy receptor Atg32. Furthermore, Atg36 binds Atg8 and the adaptor Atg11 that links receptors for selective types of autophagy to the core autophagy machinery. Atg36 delivers peroxisomes to the preautophagosomal structure before being internalised into the vacuole with peroxisomes. We conclude that Pex3 recruits the pexophagy receptor Atg36. This reinforces the pivotal role played by Pex3 in coordinating the size of the peroxisome pool, and establishes its role in pexophagy in S. cerevisiae