Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

Abstract

The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive. In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes. When PC12 cells were exposed to various extracellular K+-concentrations (to evoke depolarization-induced Ca2+ influx) or permeabilized in the presence of basal or elevated free Ca2+, levels of these SNARE complexes were altered differentially: moderate Ca2+ rises (≤1 μM) caused an increase, whereas Ca2+ elevations of more than 1 μM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged. Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.

How “Full” is “Full Fusion” during Exocytosis from Dense Core Vesicles? Effect of SDS on “Quantal” Release and Final Fusion Pore Size

“…The responses observed during successive stimulations up to the 7 th one resulted quantitatively undistinguishable from those evoked by the first one (data not shown) as evidenced qualitatively in Figure 2D showing that no significant long-term effects occurred. In agreement with previous reports, [49][50][51][52][53] this survey of SDS on exocytosis confirmed that 250-350 μM corresponded to an adequate concentration range for our purpose in this study. Yet, to ensure the absence of any non-obvious long-term effects, only the spikes delivered during the first 10 s range after the first stimulation were treated quantitatively.…”

“…42,43 Conversely, at the low concentrations used in this work SDS has been shown to have no effect on the SNAREs machineries that lead to initial fusion pores formation. 51,52 Hence, eliciting exocytosis by SDS at these low concentrations should not tamper at all with the formation of calcium-induced SNAREs complexes that initiates the critical process leading to the opening of the initial exocytotic fusion pores. Finally, it is important to note that exposing a cell to a brief pulse of low SDS concentrations cannot at all modify its vesicles internal structure nor their catecholamine content since this should require at least several minutes.…”

Characterization of SNAREs Determines the Absence of a Typical Golgi Apparatus in the Ancient Eukaryote Giardia lamblia

“…It is known that high molecular weight (HMW) bands are not detected in samples that have been boiled prior to electrophoresis. However, in Giardia, when the samples were boiled, we found some peculiarities that had not been described in other eukaryotes (59). First, some of the putative gSNAREs (gQa1-4; gR1-3) presented almost the same bands obtained in unboiled samples, suggesting that the majority of HMW bands are not heat-sensitive (supplemental Fig.…”

“…The four helices form a coiled-coil structure that in most cases is SDS-resistant at temperatures of ϳ80°C but not at boiling temperature (59). SDS-resistant SNARE complexes often exist in various forms with different electrophoretic motilities that give rise to multiple anti-SNARE-immunoreactive bands on Western blots (59).…”

“…S8). In higher eukaryotes, it is known that a property described for native SNARE complexes and for complexes formed from recombinant SNARE proteins is the temperature dependence of their SDS resistance (59). To test whether this was also true for SDSresistant complexes in Giardia, we investigated the temperature sensitivity of the higher bands found with some putative SNAREs such gQc3, gQa5, and gQb4.…”