Tomato Rab11a Characterization Evidenced a Difference Between SYP121-Dependent and SYP122-Dependent exocytosis

Abstract

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat beta-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory events.

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

“…Along with the Qa-SNARE SYP132, which is constitutively expressed and present at low levels (Enami et al, 2009), these proteins mediate secretory vesicle traffic, contributing to cellular homeostasis, expansion, and growth (Blatt, 2000;Shope et al, 2003;Campanoni and Blatt, 2007). Functional differences between SYP121 and SYP122 are known (Leyman et al, 1999;Zhang et al, 2007;Rehman et al, 2008;Honsbein et al, 2009Honsbein et al, , 2011Grefen et al, 2010a;Eisenach et al, 2012), but as these Qa-SNAREs share cognate partners Karnik et al, 2013b;Yun et al, 2013), studies to date have offered few clues to the mechanistic basis for the differences in their regulation and function. Among other factors, SM proteins are widely recognized to be key components that regulate SNARE-mediated vesicle traffic, both in stabilizing the cognate SNARE complex and in controlling the availability of several Qa-SNAREs for binding (Südhof and Rothman, 2009;Rizo and Südhof, 2012).…”

“…The Qa-SNAREs of the plasma membrane are a case in point. In Arabidopsis thaliana, two of these SNAREs, SYP121 (=SYR1/PEN1) and SYP122, show a high degree of structural homology and overlap in function (Assaad et al, 2004;Tyrrell et al, 2007;Bassham and Blatt, 2008;Rehman et al, 2008). Single mutations of these Qa-SNAREs have little effect on plant growth under many conditions, and reduced growth is normally evident only in the syp121/syp122 double mutant (Assaad et al, 2004;Zhang et al, 2007).…”

“…Both SYP121 and SYP122 are expressed throughout the development of the plant and in most, if not all, tissues (Uemura et al, 2004;Enami et al, 2009). The Qa-SNAREs also share the cognate partner proteins SNAP33, VAMP721, and VAMP722 Rehman et al, 2008;Karnik et al, 2013b), indicating substantial mechanistic overlaps in driving vesicle fusion. Nonetheless, a number of functional differences between SYP121 and SYP122 have surfaced.…”

Nicotiana tabacumprotoplasts secretome can evidence relations among regulatory elements of exocytosis mechanisms

“…1 Monitoring an exogenous reporter called secRGUS, it was possible to distinguish between SYP121 and SYP122 DN effects. The analysis of an exogenous marker, transiently expressed by the cells, certainly simplify the approach to this peculiar topic, where many genes appear to have functional redundancy just because a valid assay of their specificity is not available.…”

Arabidopsis TRAPPII is functionally linked to Rab-A, but not Rab-D in polar protein trafficking intrans-Golgi network

“…[6][7][8][10][11][12][13][14] For example, roles of small Rab-A GTPases, homologs of yeast Ypt31/32 and animal Rab11 in TGN trafficking to the cell plate in cytokinesis 10 and in polar secretion of cell wall components 6,7,13 have been demonstrated. Using a combined approach of genetic and in vivo imaging, we recently shown that Arabidopsis TRAPPII acts in intracellular trafficking in the TGN in root tip cells.…”