GENE FUSIONS OF SIGNAL SEQUENCES WITH A MODIFIED BETA-GLUCURONIDASE GENE RESULTS IN RETENTION OF THE BETA-GLUCURONIDASE PROTEIN IN THE SECRETORY-PATHWAY PLASMA-MEMBRANE

Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secr etory pathway, but molecular aspects about their involvement are poorl y understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phase olus vulgaris; Serpin from Manduca sexta) to direct a modified beta-gl ucuronidase (GUS) protein into the secretory pathway in transgenic tob acco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular flu id analysis (low-or high-ionic-strength conditions) but occurred in me mbranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equili brated with the plasma membrane (PM) and the ER on linear sucrose grad ients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed c ultured cells prepared from GUS control and HRGP signal peptide (SP)-G US-expressing plants suggested only cytosolic localization in GUS-expr essing plants but substantial peripheral localization in HRGP SP-GUS p lants, which is consistent with GUS being associated with the PM. Aque ous two-phase partitioning of microsomal membranes from HRGP SP-GUS an d Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydr ophobic moment plot analysis, suggest that properties of the SP-GUS pr otein result in its retention in the secretory pathway and PM.