Peroxisome matrix and membrane protein biogenesis.

Peroxisomes play an important role in lipid metabolic pathways and are implicated in many human disorders. Their biogenesis has been studied over the last two decades using many uni and multi-cellular model systems and many aspects of the mechanisms and proteins involved in peroxisome biogenesis are conserved from yeast to humans. In this manuscript we review the recent progress made in our understanding of the mechanisms by which matrix and membrane proteins are sorted to and assembled into peroxisomes.

An in vitro study on human ureteric smooth muscle with the alpha1-adrenoceptor subtype blocker, tamsulosin.

Objective: To study the effects of tamsulosin on ureteric contractions and its effects on the basal tone of human ureteric specimens, as clinical trials with tamsulosin have shown promising results in the spontaneous expulsion of lower ureteric calculus, but the mechanism of action of tamsulosin in the expulsion of ureteric calculus has not been elucidated in in-vitro studies on human ureters.

Materials And Methods: Human mid-ureteric specimens were obtained from live kidney donors. The specimen was transported in Krebs' solution and the isometric contraction of human ureteric smooth muscle was recorded in the presence of tamsulosin.

Knowledge of diabetes and diabetic retinopathy among rural populations in India, and the influence of knowledge of diabetic retinopathy on attitude and practice.

Introduction: Diabetes mellitus, particularly type II, is a major public health concern worldwide. While the occurrence of diabetic retinopathy cannot be prevented, with the provision of knowledge to sufferers, sight-threatening complications can be minimized.

Purpose: To report the results of a KAP (Knowledge, Attitude and Practice) study among a rural population in two areas: diabetes mellitus (DM) and diabetic retinopathy (DR).

Special delivery from mitochondria to peroxisomes.

Inter-organellar communication and interactions are necessary and accepted consequences of the segregation of biochemical functions into subcellular organelles. Recently, Heidi McBride and her collaborators found a novel link between mitochondria and peroxisomes in their discovery of mitochondria-derived vesicles (MDVs), which appear to fuse with a fraction of pre-existing peroxisomes in mammalian cells. We discuss the potential role of this vesicle population in the context of pathways for the exchange of metabolites and/or macromolecules between these compartments.

PpAtg30 tags peroxisomes for turnover by selective autophagy.

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy.

Dysferlin domain-containing proteins, Pex30p and Pex31p, localized to two compartments, control the number and size of oleate-induced peroxisomes in Pichia pastoris.

Yarrowia lipolytica Pex23p and Saccharomyces cerevisiae Pex30p, Pex31p, and Pex32p comprise a family of dysferlin domain-containing peroxins. We show that the deletion of their Pichia pastoris homologues, PEX30 and PEX31, does not affect the function or division of methanol-induced peroxisomes but results in fewer and enlarged, functional, oleate-induced peroxisomes. Synthesis of Pex30p is constitutive, whereas that of Pex31p is oleate-induced but at a much lower level relative to Pex30p.

Localization of proteins and organelles using fluorescence microscopy.

This chapter describes the different methods used for localization of proteins and organelles in Pichia pastoris. A series of plasmids and a modified immunofluorescence protocol for localization and co-localization of proteins and organelles are described. Also included are protocols for the labeling of different subcellular organelles with vital stains.

Characterization of protein-protein interactions: application to the understanding of peroxisome biogenesis.

With the approaching completion of the Pichia pastoris genome, a greater emphasis will have to be placed on the proteome and the protein-protein interactions between its constituents. This chapter discusses methods that have been used for the study of such interactions among both soluble and membrane-associated proteins in peroxisome biogenesis. The procedures are equally applicable to other cellular processes.

A ubiquitin-like protein involved in membrane fusion.

Atg8 is a ubiquitin-like protein involved in autophagy in yeast that is targeted to membranes through conjugation to the lipid phosphatidylethanolamine (PE). In this issue of Cell, Nakatogawa et al. (2007) show that Atg8 conjugated to PE mediates tethering between adjacent membranes and stimulates membrane hemifusion, an event that may mimic expansion of the autophagosomal membrane during autophagy.

Autophagy-related pathways and specific role of sterol glucoside in yeasts.

Recently, we showed that the requirement of sterol glucoside (SG) during pexophagy in yeasts is dependent on the species and the nature of peroxisome inducers. Atg26, the enzyme that converts sterol to SG, is essential for degradation of very large methanol-induced peroxisomes, but only partly required for degradation of smaller-sized oleate- and amine-induced peroxisomes in Pichia pastoris. Moreover, oleate- and amine-induced peroxisomes of another yeast, Yarrowia lipolytica, are degraded by an Atg26-independent mechanism.

A cytoplasm to vacuole targeting pathway in P. pastoris.

The cytoplasm-to-vacuole targeting (Cvt) pathway of Saccharomyces cerevisiae delivers aminopeptidase I (Ape1) from the cytosol to the vacuole, bypassing the normal secretory route. The Cvt pathway, although well-studied, was known only in S. cerevisiae.

A conserved cysteine residue of Pichia pastoris Pex20p is essential for its recycling from the peroxisome to the cytosol.

We identified a cysteine residue, conserved near the N terminus of Pex5p- and Pex20p-like proteins, that is essential for the cytosolic relocation of peroxisomal Pex20p. Surprisingly, this residue is not completely essential for the function of the protein; its point mutation into a serine in Pex20p(C8S) causes the accumulation of the protein at the peroxisome membrane, but this is quickly followed by its subsequent degradation by an ubiquitin-dependent quality control pathway called RADAR (receptor accumulation and degradation in the absence of recycling). This degradative pathway allows partial growth of the Pex20p(C8S) mutant on peroxisome-requiring medium.

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers.

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy.

Calcium-transporters in myocardial cells.

This is a concise review of important calcium-transporters on the sarcolemma and organellar membranes of myocardial cells, and their functional roles in cell physiology. It briefly addresses L and T type calcium channels, store-operated calcium channel (SOC), sodium-calcium exchanger (NCX), and the plasma membrane calcium ATPase (PMCA) on the sarcolemma, ryanodine receptor (RyR), IP3 receptor (IP3R) and the sarcoplasmic reticulum (SR) calcium ATPase (SAERCA) on the SR membrane and their contributions to contraction and rhythm-generation. Several agonists and blockers for every transporter that are commonly used in research, and those with therapeutic applications have also been discussed.

The importomer--a peroxisomal membrane complex involved in protein translocation into the peroxisome matrix.

The import of proteins into the peroxisome matrix is an essential step in peroxisome biogenesis, which is critical for normal functioning of most eukaryotic cells. The translocation of proteins across the peroxisome membrane and the dynamic behavior of the import receptors during the import cycle is facilitated by several peroxisome-membrane-associated protein complexes, one of which is called the importomer complex [B. Agne, N.

Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors.

Based on earlier suggestions that peroxisomes may have arisen from endosymbionts that later lost their DNA, it was expected that protein transport into this organelle would have parallels to systems found in other organelles of endosymbiont origin, such as mitochondria and chloroplasts. This review highlights three features of peroxisomal matrix protein import that make it unique in comparison with these other subcellular compartments - the ability of this organelle to transport folded, co-factor-bound and oligomeric proteins, the dynamics of the import receptors during the matrix protein import cycle and the existence of a peroxisomal quality-control pathway, which insures that the peroxisome membrane is cleared of cargo-free receptors.

Atg28, a novel coiled-coil protein involved in autophagic degradation of peroxisomes in the methylotrophic yeast Pichia pastoris.

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy. We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris.

Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.

Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate.

Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation.

We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p.

Two independent pathways traffic the intraperoxisomal peroxin PpPex8p into peroxisomes: mechanism and evolutionary implications.

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix.

Force-frequency relation in frog-ventricle is dependent on the direction of sodium/calcium exchange in diastole.

Aim: Force of contraction increases with stimulus-frequency in mammalian and amphibian hearts under control conditions. Here, we have analysed the mechanism of the force-frequency relation (FFR) in frog-ventricle.

Methods: Circular strips of frog-ventricle were subjected to field-stimulation with frequencies in the range 0.

The control of peroxisome number and size during division and proliferation.

Like other subcellular organelles, peroxisomes divide and segregate to daughter cells during cell division, but this organelle can also proliferate or be degraded in response to environmental cues. Although the mechanisms and genes involved in these processes are still under active investigation, an important player in peroxisome proliferation is a dynamin-related protein (DRP) that is recruited to the organelle membrane by a DRP receptor. Related DRPs also function in the division of mitochondria and chloroplasts.

Peroxisome turnover by micropexophagy: an autophagy-related process.

Many organisms stringently regulate the number, volume and enzymatic content of peroxisomes (and other organelles). Understanding this regulation requires knowledge of how organelles are assembled and selectively destroyed in response to metabolic cues. In the past decade, considerable progress has been achieved in the elucidation of the roles of genes involved in peroxisome biogenesis, half of which are affected in human peroxisomal disorders.