Sorting of GFP tagged NtSyr1, an ABA Related Syntaxin

Abstract

Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.

Expression of a glycosylated GFP as a bivalent reporter in exocytosis

“…This reporter may be used to study protein sorting in different species with a comparative approach (Di Verweij et al 2008) as well as transmembrane protein topology (Di Sansebastiano et al 2006).…”

“…It was recently renamed 121T for clarity (Di Sansebastiano et al 2006;Rehman et al 2008), to be distinguished from other SYPs mutants.…”

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

“…The truncated forms of SYPs (121T and 122T), deprived of the trans-membrane domain at the C-terminus, 1,19 as well as the H3 forms, where the N-terminus was substituted by GFP, 20 were previously described. Here for the first time we tested the effect of a truncated form of SNAP33 (SNAP33D).…”

“…"H3" versions 20 of SYP121 (121H3) and SYP122 (122H3) were used directly to test the effect on a larger set of secreted proteins, and compared to reveal how similar these domains of SYPs could be.…”

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: Engineering attempts using a modified SNARE protein

“…SYP51 seems to be trapped on a membrane where it should just transit. Other H3 mutants have been prepared in the past for other SNARE proteins and induced aberrant structures (Di Sansebastiano et al, 2006), anyhow in this case this structure represents an interruption of traffic and, apparently, not a diversion to a wrong final destination. If the small vacuoles induced by GFP:At51H3 have not the same characteristics of the central vacuole, generally lytic, they may maintain their content more stable, so providing an advantage in the accumulation and stability of interesting products.…”