TY - JOUR
T1 - Selective targeting of plasma membrane and tonoplast traffic by inhibitory (dominant-negative) SNARE fragments
AU - Tyrrell, Matthew
AU - Campanoni, Prisca
AU - Sutter, Jens Uwe
AU - Pratelli, Réjane
AU - Paneque, Manuel
AU - Sokolovski, Sergei
AU - Blatt, Michael R.
PY - 2007/9
Y1 - 2007/9
N2 - Vesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1-YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants.
AB - Vesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1-YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants.
KW - Arabidopsis thaliana
KW - Endoplasmic reticulum
KW - Membrane vesicle traffic
KW - Nicotiana tabacum
KW - Secreted GFP
KW - Tonoplast intrinsic protein
UR - http://www.scopus.com/inward/record.url?scp=34548504555&partnerID=8YFLogxK
UR - https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-313X.2007.03206.x
U2 - 10.1111/j.1365-313X.2007.03206.x
DO - 10.1111/j.1365-313X.2007.03206.x
M3 - Article
C2 - 17662029
AN - SCOPUS:34548504555
SN - 0960-7412
VL - 51
SP - 1099
EP - 1115
JO - Plant Journal
JF - Plant Journal
IS - 6
ER -