Poster: Organelle biogenesis

Abs # 629: The Tomato Bushy Stunt virus replicase protein p33 contains multiple peroxisomal membrane targeting signals and causes the formation of novel vesicles adjacent to aggregated peroxisomes

Presenter:	McCartney, Andrew W., amccar01@uoguelph.ca
Authors	McCartney, Andrew W. (A)   Greenwood, John S. (A)   Mullen, Robert T. (A)
Affiliations:	(A): Department of Botany, University of Guelph

Tomato Bushy Stunt virus(TBSV) is a single-stranded RNA virus that encodes five proteins including a membrane-bound replicase protein, termed p33. Infection of plant cells with TBSV leads to progressive invaginations of the peroxisomal boundary membrane and eventually the formation of an altered peroxisome known as a multivesicular body (MVB), the proposed site of TBSV RNA replication. While the origin of MVBs is known to be due, in part, to p33, the molecular mechanisms involved in the sorting of nascent p33 to peroxisomes or the remodelling of peroxisomal membranes during MVB formation are not known. Thus, in order to gain a better understanding of MVB biogenesis, and perhaps peroxisome biogenesis in general, we characterized the membrane peroxisomal targeting signals (mPTSs) within p33 using site-directed mutagenesis and tobacco BY-2 suspension cells as a transient expression system. Overall, three distinct mPTSs were identified within p33. Interestingly, p33 expressed alone in either BY-2 cells or tobacco leaf cells caused dramatic alterations in peroxisomal distribution, e.g., aggregation, but not invaginations of peroxisomal boundary membranes, such as those observed during normal MVB formation in TBSV-infected cells. Instead, aggregated peroxisomes in p33-expressing cells were surrounded by numerous, small vesicles that contained the replicase protein. Whether these novel vesicles were derived from (proliferated) peroxisomal membranes following the targeting and insertion of p33 or originated from another endomembrane source (ER?) is currently being examined. Supported by NSERC grant 217291 to RTM.