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We recently identified a novel and transplantable di-acidic motif (EXXD) that facilitates ER export of the Golgi syntaxin SYP31 (type IV protein) and which may function also for type I and type II proteins in plants. By mutagenesis of Arabidopsis thaliana SYP31 and live cell imaging experiments in tobacco leaf epidermal cells, we determined that replacing the MELAD sequence of SYP31 with gagag retained SYP31 in the ER, which demonstrates that the di-acidic motif ELAD is critical for SYP31 ER export. To investigate whether blockage of a Golgi SNARE in the ER have consequences for plant growth, we produced tobacco plants stably overexpressing either the wild type MELAD or the mutant gagag form of SYP31. Whereas tobacco plants overexpressing the wild-type SYP31 developed to set seed, tobacco plants overexpressing the mutant form gagag rapidly became chlorotic, ceased their growth and invariably died after several weeks. This indicated that retention of overexpressed SYP31 in the ER is likely toxic for the secretory pathway and, therefore, plant development. Putative explanations for this observation are discussed taking into account SNARE properties and possible interactions.
SNAREs (soluble N-ethyl-maleimide sensitive factor attachment receptor proteins) are components of the molecular machinery that facilitates vesicular transport in the secretory pathway of eukaryotic cells,1,2 and are critical for numerous plant physiological functions.1,3 SYP31 is a type IV syntaxin localized at the Golgi and it is required for anterograde traffic from the ER to the Golgi.4,5 In the search for putative ER export signals in the sequence of SYP31, we identified a di-acidic motif (M) ELAD(G) of the type EXXD.6 This di-acidic motif was essential for ER export and Golgi targeting of SYP31, and we suggested an interaction of this motif with the COPII machinery (reviewed in ref. 6). To investigate whether blocking a Golgi SNARE in the ER may affect plant growth, we have produced transgenic tobacco plants overexpressing either the wild type MELAD or the mutant gagag form of A. thaliana SYP31.
Assays to obtain stable transgenic tobacco plants were performed following the procedure described by Sparkes et al.7 The infiltrated leaf sections were placed in shooting media and resistant shoots were transferred to the same shooting media but without growth regulators (rooting media). Every 3–4 weeks, the growing plants were multiplied by sub-culturing to new Petri dishes containing rooting media. Two week-old in vitro grown plants expressing either the wild type SYP31-YFP construct or the mutant form gagag are shown in Figure 1. Using this approach we were able to obtain seeds from stably transformed tobacco plants for the wild type SYP31-YFP construct (Fig. 1A). In contrast, plants expressing the mutant form gagag of SYP31 never progressed beyond three months of subculture, as the plants rapidly became chlorotic and arrested their growth, which made their multiplication difficult. We made three separate attempts to produce stably transformed plants with the mutant form gagag of SYP31, but all attemps were unsuccessful due to the systematic strong reduction in the growth of the plants (Fig. 1B). Plants expressing either the wild-type SYP31 or the mutant form gagag produced similar amounts of protein product as verified by measuring protein amounts from isolated microsomal membranes (Fig. 1C). The impossibility to produce stable transgenic plants with the mutant gagag form of SYP31 strongly supports the idea that the retention of SYP31 in the early secretory pathway is highly toxic and therefore deleterious for plant growth.
It is possible that the accumulation of a SNARE in a different compartment could affect the steady-state behavior of putative partners. Miller et al.8 suggested that one of the roles of the yeast SM protein Sly1 (Sec1/Munc18-like protein) is to interact with Sed5 (homolog to SYP31) in the ER, in order to maintain Sed5 in a conformation that leaves the ER export motif NPF of Sed5 accessible to Sec24 (COPII machinery). Therefore, the retention of SYP31 in the ER may disturb the homeostasis of Sly1. Alternatively, it has been proposed that trafficking of ER and Golgi SNAREs from the ER as individual proteins may be a general mechanism in eukaryotes.10 The packaging of SNAREs into COPII vesicles independent of SNARE pairing8–10 may be to isolate a functional SNARE until it reaches its appropriate membrane, where it performs its normal function. Therefore, we may also suggest that retention and/or accumulation of functional SYP31 in the ER could induce uncontrolled SNARE interactions, enough to trap the interacting SNAREs or their partners in the ER. As a consequence, other normal SNARE complexes that arise from their combinatorial specificity11 would also not be produced. It has been proposed that these complex SNARE pairings contribute to maintain the compartmental organization and identity of membranes of the secretory pathway.12 Therefore, retention and/or accumulation of SYP31 in the ER may greatly impair the targeting of other SNAREs and/or the formation of specific SNARE complexes resulting in overall malfunction of the secretory pathway, and therefore in defects in cell growth and plant development.
It has been suggested also that nonfunctional SNARE complexes may have a physiological relevance, and the so called “i-SNAREs” (inhibitory SNAREs) may contribute to control the specificity of membrane fusion.13 Depending on the SNARE concentration gradients in the Golgi, SNAREs may have a normal SNARE function or an i-SNARE function in different cisternae, enough to create a buffering effect on the capacity of vesicles to fuse in a given cisternae.13 In our case, retention of SYP31 in the ER could induce formation of abnormal SNARE complexes in this compartment, and as a consequence, a constitutive or unregulated “i-SNARE” toxic effect against the correct functioning of the ER-Golgi interface may take place with detrimental effects to plant growth.
Finally, we may also consider that retaining SYP31 in the ER could affect the maintenance of distinctive ER and Golgi interfaces since the morphology of the Golgi is known to be linked to Sed5 (SYP31 homolog) phosphorylation in yeast.14 The perturbations in the homeostasis of the formation of specific SNARE complexes and the potential strong changes in the morphology/identity of ER and Golgi membranes may additively or independently help explain the observed deleterious effects on plant development upon retention of the SNARE SYP31 in the ER.
Previously published online: www.landesbioscience.com/journals/psb/article/9643