Expression of the Cymbidium Ringspot Virus 33-Kilodalton Protein in Saccharomyces cerevisiae and Molecular Dissection of the Peroxisomal Targeting Signal

Abstract

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

“…Alfalfa mosaic virus and cucumber mosaic virus (both of the Bromoviridae) replicate in association with tonoplast membrane (Van Der Heijden et al, 2001;van der Heijden & Bol, 2002;Sztuba-Solinska & Bujarski, 2008). Most members of the Tombusviridae studied to date replicate in association with peroxisome membranes (Navarro et al, 2004(Navarro et al, , 2006McCartney et al, 2005;Panavas et al, 2005;Jonczyk et al, 2007). Turnip yellow mosaic virus (TYMV, family Tymoviridae) replicates on chloroplast outer membrane (Prod'homme et al, 2001).…”

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

“…Protocols for partial purification and agarose separation of whole virus particles described by Crivelli and associates (2011) were used, with some modification. Briefly 1 g of agroinfiltrated leaves was homogenized in 8 ml of protein extraction buffer (Navarro et al 2004) and incubated at 37°C for 30 min. The extract was then mixed with an equal volume of chloroform and centrifuged at 3,000 × g for 10 min.…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…(C) Proliferación de peroxisomas en células de levadura que expresan la proteína auxiliar de la replicación (p33) del virus de las manchas anulares del Cymbidium (CymRSV). El marcaje muestra la proteína situada principalmente en las membranas peroxisomales [Navarro et al, 2004]. (D) Localización de la RdRp (proteína 2a) del virus del mosaico del pepino (CMV) en las vesículas del tonoplasto vacuolar (v) en una sección transversal de un vaso criboso (sieve element, SE) en hojas de tabaco infectadas.…”

“…La información disponible actualmente acerca de las replicasas de los miembros de la familia Tombusviridae se ha obtenido fundamentalmente a partir de los trabajos realizados con varios miembros del género Tombusvirus [Bleve-Zacheo et al, 1997;Oster et al, 1998;Rubino et al, 2001;Weber-Lotfi et al, 2002;Panaviene et al, 2003;Navarro et al, 2004;Panavas et al, 2005a;] y con una especie del género Dianthovirus [Bates et al, 1995;Turner et al, 2004;Takeda et al, 2005;Okamoto et al, 2008;Mine et al, 2010b]. Estas investigaciones se han centrado en los requerimientos de secuencias y las actividades enzimáticas de la RdRp, en las propiedades de unión a ácidos nucleicos de ambas replicasas, su localización subcelular, la búsqueda de factores del huésped implicados en la replicación y en la interacción entre ambas proteínas.…”

“…Entre los miembros del género Tombusvirus, se ha descrito la asociación de la proteína p33 del TBSV, el CymRSV y el CNV a membranas peroxisomales [Navarro et al, 2004;Panavas et al, 2005a;McCartney et al, 2005], mientras que la proteína homóloga del CIRV, p36, se localiza en mitocondrias [Rubino et al, 2000. Tanto la proteína p33 como la proteína p36 inducen la formación de múltiples vesículas esféricas u ovoides (MVB) resultado de la invaginación de la membrana externa del orgánulo con el que se asocian.…”