Biogenesis of Glyoxysomes

Biosynthesis of isocitrate lyase, a tetrameric enzyme of the glyoxysomal matrix, was studied in Neurospora crassa, in which the formation of glyoxysomes was induced by a substitution of sucrose medium by acetate medium. * 1. Translation of Neurospora mRNA in reticulocyte lysates yields a product which has the same apparent molecular weight as the subunit of the functional enzyme. Using N-formyl[35S]methionyl tRNAMetf as a label, the translation product shows the same apparent size which indicates that the amino terminus has no additional 'signal'-type sequence. * 2. Read-out systems employing free and membrane-bound polysomes show that only free ribosomes are active in the synthesis of isocitrate lyase. * 3. Isocitrate lyase synthesized in reticulocyte lysate is released into the supernatant and is soluble in a monomeric form. It interacts with Triton X-100 to form mixed micells in contrast to the functional tetrameric form. * 4. Transfer of isocitrate lyase synthesized in vitro into isolated glyoxysomes is suggested by results of experiments in which supernatants from reticulocyte lysates are incubated with a particle fraction isolated from acetate-grown cells. No transfer occurs when particles from non-induced cells are employed. Resistance to added proteinase is used as a criterion for transmembrane transfer. The data support a post-translational transfer mechanism for isocitrate lyase. They suggest that isocitrate lyase passes through a cytosolic precurscr pool as a monomer and is transferred into glyoxysomes

Harmonic vs. subharmonic patterns in a spatially forced oscillating chemical reaction

The effects of a spatially periodic forcing on an oscillating chemical reaction as described by the Lengyel-Epstein model are investigated. We find a surprising competition between two oscillating patterns, where one is harmonic and the other subharmonic with respect to the spatially periodic forcing. The occurrence of a subharmonic pattern is remarkable as well as its preference up to rather large values of the modulation amplitude. For small modulation amplitudes we derive from the model system a generic equation for the envelope of the oscillating reaction that includes an additional forcing contribution, compared to the amplitude equations known from previous studies in other systems. The analysis of this amplitude equation allows the derivation of analytical expressions even for the forcing corrections to the threshold and to the oscillation frequency, which are in a wide range of parameters in good agreement with the numerical analysis of the complete reaction equations. In the nonlinear regime beyond threshold, the subharmonic solutions exist in a finite range of the control parameter that has been determined by solving the reaction equations numerically for various sets of parameters.Comment: 14 pages, 11 figure

Kinetic Studies on the Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in Intact Cells of Neurospora crassa

The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques. In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins. The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics. Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria. Mitochondrial matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship. The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondrial pool from which they are imported into the mitochondria

Pattern orientation in finite domains without boundaries

We investigate the orientation of nonlinear stripe patterns in finite domains. Motivated by recent experiments, we introduce a control parameter drop from supercritical inside a domain to subcritical outside without boundary conditions at the domain border. As a result, stripes align perpendicular to shallow control parameter drops. For steeper drops, non-adiabatic effects lead to a surprising orientational transition to parallel stripes with respect to the borders. We demonstrate this effect in terms of the Brusselator model and generic amplitude equations

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m- chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane

Migration reversal of soft particles in vertical flows

Non-neutrally buoyant soft particles in vertical microflows are investigated. We find, soft particles lighter than the liquid migrate to off-center streamlines in a downward Poiseuille flow (buoyancy-force antiparallel to flow). In contrast, heavy soft particles migrate to the center of the downward (and vanishing) Poiseuille flow. A reversal of the flow direction causes in both cases a reversal of the migration direction, i. e. heavier (lighter) particles migrate away from (to) the center of a parabolic flow profile. Non-neutrally buoyant particles migrate also in a linear shear flow across the parallel streamlines: heavy (light) particles migrate along (antiparallel to) the local shear gradient. This surprising, flow-dependent migration is characterized by simulations and analytical calculations for small particle deformations, confirming our plausible explanation of the effect. This density dependent migration reversal may be useful for separating particles.Comment: 8 pages, 7 figure