A Multigene Family Encoding R‐SNAREs in the Ciliate Paramecium tetraurelia

Abstract

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division. Intracellular vesicle trafficking between different organelles, as well as to and from the cell surface, is a hallmark of eukaryotic cells. These membrane interactions are tightly regulated, and the molecular machinery mediating these events is the subject of intense current research. Although many details are similar, some are highly divergent in different cells or from one membrane system to another. We are particularly interested in such aspects in the ciliated protozoan Paramecium tetraurelia that possesses elaborate membrane-trafficking systems (1). Membrane interactions, such as the fusion of intracellular vesicles with other vesicles or the cell membrane, are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) which are characterized by a heptad repeat coiled-coil SNARE motif (2-4). SNARE proteins mediate membrane interactions and have been classified as target-SNAREs (t-SNAREs) and vesicle-SNAREs (v-SNAREs) according to their localization (5). Before membranes fuse, v-SNAREs and t-SNAREs in the opposing membranes form a stable trans-SNARE complex with their coiled-coil SNARE domains that is thought to be prerequisite to bring about fusion of the vesicle with the target membrane (6-8). Because SNARE pairing is specific to different membrane-trafficking pathways, cognate pairing of individual v-SNAREs with corresponding members of the large family of t-SNAREs is required before fusion can occur (4,6,7). This is reflected by the distinct subcellular localizations of different SNARE isoforms (4). One of the best characterized SNARE complexes is the synaptic vesicle SNARE-pin, formed by the t-...

An Elaborate Classification of SNARE Proteins Sheds Light on the Conservation of the Eukaryotic Endomembrane System

“…In subsequent studies, the presence of different conserved syntaxin subfamilies (i.e., mostly Qa-SNAREs) in several diverse eukaryotes supported the notion that these SNAREs diverged early in eukaryotic evolution Doolittle, 2002, 2004). The early diversification of SNARE proteins is also corroborated by the fact that other, distantly related eukaryotes encompass relatively conserved SNARE sets (Besteiro et al, 2006;Schilde et al, 2006;Yoshizawa et al, 2006;Ayong et al, 2007;Kissmehl et al, 2007;and our analysis). Notably, our study substantiates the view that all SNARE proteins principally split into the four major phylogenetic classes: Qa, Qb, Qc, and R (Bock et al, 2001), suggesting that the prototypic unit was composed of four different SNARE proteins, able to assemble into a tight fourhelix bundle between two fusing membranes.…”

“…Similarly, SNAREs of the kinetoplastid parasites Trypanosoma brucei, T. cruzi, and Leishmania major usually also congregated, reflecting their close relationship. In most protists with entire genomes available, we discovered a reasonable collection of SNARE proteins, supplementing the so far established repertoires Doolittle, 2002, 2004;Besteiro et al, 2006;Schilde et al, 2006;Yoshizawa et al, 2006;Ayong et al, 2007;Kissmehl et al, 2007). For example, for Plasmodium falciparum we found 22 SNAREs with almost all different subgroups represented.…”

“…In addition, the original classification of SNARE proteins (Bock et al, 2001) was based on only ϳ100 sequences from only few organisms and did not include some more diverged SNARE types (Lewis et al, 1997;Lewis and Pelham, 2002;Burri et al, 2003;Dilcher et al, 2003). In the subsequent years, additional complete genomes shed more light onto the conservation of the SNARE machinery, yet some SNAREs, in particular from protists, appeared to be rather atypical (Sanderfoot et al, 2000; Doolittle, 2002, 2004;Gupta and Brent Heath, 2002;Uemura et al, 2004;Besteiro et al, 2006;Schilde et al, 2006;Sutter et al, 2006;Ayong et al, 2007;Kissmehl et al, 2007;Sanderfoot, 2007). These studies, however, did not provide a universal classification scheme, as usually only a subset of sequences was examined.…”

Evolution of the eukaryotic membrane-trafficking system: origin, tempo and mode

“…Functional characterization in the amoebae (Bogdanovic et al, 2002;Nakada-Tsukui et al, 2005), chromalveolates such as Toxoplasma, Plasmodium and the ciliates (Ngo et al, 2003;Schilde et al, 2006;Struck et al, 2005), excavates such as kinetoplastids (Besteiro et al, 2006;Morgan et al, 2002) and Giardia (Lujan and Touz, 2003) and plants further support the idea that the LCEA had a complex membrane trafficking system. These studies have several implications for our understanding of the biology of membrane trafficking.…”

The SEC6 protein is required for function of the contractile vacuole inChlamydomonas reinhardtii

“…Despite this structural diversity the basic functions seem to be conserved between different eukaryotes because the same proteins and cellular processes have been found in Amoeba, Dictyostelium, Paramecium, Trypanosoma and green algae [e.g. V-ATPase (Becker and Hickisch, 2005;Fok et al, 2002;Heuser et al, 1993;Montalvetti et al, 2004;Nishihara et al, 2008;Robinson et al, 1998;Wassmer et al, 2005), aquaporin Nishihara et al, 2008), vesicular transport (Becker and Hickisch, 2005;Buchmann and Becker, 2009;Bush et al, 1994;Harris et al, 2001;Kissmehl et al, 2007;Schilde et al, 2006;Stavrou and O'Halloran, 2006); see KomsicBuchmann and Becker for a summary of identified proteins and cellular processes (Komsic- Buchmann and Becker, 2012)]. …”