Specific aspects of SNAREs in Paramecium

The aspects just discussed are particularly interesting if one considers the longin character of R-SNAREs in P. tetmurelia, including members of the PtSybl, 2, 3, and 6-9 subfamilies (Schilde et al., 2006, 2(10) as well as PtSec22 (Kissmehl et al., 20CO). The question arises as to similar effects of the longin domain (Section 3.1. 1), including targeting, in plants and in Pammecium-a question to be analyzed in future work. In Fig. 3.3, we give an example what Paramecium SNAREs look like and where they are loca-lized. As 'T'ables 3.1 and 3.2 shm'V, some of the Paramecium SNAREs display various aberrant features. This may include absence of a transmembrane domain (PtSyb6 and 7), substitution of the R-residue in some PtSybforms (PtSyb8-11) or of the Q-residue in one of the PtSyTx molecules (PtSyx12).

(N ote that such aberrations also occur in established higher eukaryotic

systems [Fasshauer et aI., 1998]). Even a SNARE domain may not be identifiable in some of the Paramecium R-SNAREs, such as PtSyb4 and 5 which are prognosticated as SNAREs by overall homology (Table 3 '1).

Although they display distinct subcellular localization there may be func-tional implications yet to be analyzed. The expected lipidic anchor seems to be absent fi~om PtSNAP-25-LP (Schilde et a1., 2()08). On the one hand, its abundance in the cytosol (apart from association with niany membranes of trafficking compartments) and on the other hand the presence of other characteristic features mavJ' ustif\r the inclusion of these molecules in the list _{J J} of PtSNAREs. In fact, there are comparable examples in other cells.

Huwever, what may the absence of important features imply in fimc-tional terms-may such SNAREs be fimcfimc-tional? For the following reasons, it appears premature at this time to appreciate any role for the truncated SNAREs we found in Paramecium. (i) In yeast, the non-NSF type cocha-perone, Sec 17 (difierent fi'om the NSF homolog Sec 18) can complete fusion with normally nonfusogenic trans-SNARE complexes (Schvvartz and lVIerz, 20(9). (ii) Fragments of synaptobrevin can reduce the formation of dead-end syntaxin/SNAP-25 complexes (Pol::lbati et a1., 2CHJ6).

(iii) Soluble SNAREs can associate, in yeast, with a SNARE complex and thus drive va.cuole interaction and fusion Cfhorngren et aI., 20(4). Right away one would rather envisage some inhibitory efiect in the latter two cases. (iv) In contrast to these situations, inhibitory SNAREs have been identified in mammalian cells as a set of t-SNAREs endowed with a transmembrane domain and occurring in addition to "normal" t-SNAREs;

therefore, they may serve fine-tTming (VarIarnov et aI., 20(4). Although no such analyses have been executed with ciliates, the examples clearly indicate that absence of a transmembraTle domain ,;vould not necessarily entail an inhibitOlyl competitive role for SNAREs lacking a transmembrane segment.

Substitution of Q for R in the O-layer of SNAREs in yeast reduces cell growth and protein secretion, but can be restored by an inverse substitution in a partner SNARE (GraC et al., 2005; Ossig et al., 20(0). Similarly, a Q --+ R substitution in Syb of synaptic vesicles has no dramatic effect in hippocampal neurons (f )(:ak et a1) 20(6). In hct, deviations tiom the orthodox O-layer have been detected even in normal cells (Fasshauer et aL, 1998; Sultan et aL, 1998). However, the effect of deviating O-layer aminoacids other than Rand Q in P. tetraurelia is difficult to anticipate without detailed analysis. Deviations from the orthodox heptad repeat structure in quite a few PtSNAREs (Kissniehl et aI., 2007; Schilde et a1., 2006, 2010; 'rables 3.1 and 3.2) may reduce SNARE specificity (Fasshauer et al., 1998; Graf et ~ll.) 2005; Paumet et al , 20(4) and zippering, and, thus, fusogenicity.

In conclusion, we have identified numerous SNAREs, type R, Qa, Qc, Qbl c, in P. tetraurelia, cloned the respective genes, localized the proteins, and largely probed their function by gene silencing (as discussed in

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subsequent sections). These are the only data available on SNAREs in ciliates. Their number is about rvvice that assumed for the ur-eukaryote particularly vvhen one also considers the twin isoforms originating from a recent whole genOlne duplication ("ohnologs"). These may now mainly serve gene amplification, in order to match the requirements for intense vesicle trafficking. A substantial number of PtSNAREs contributes to the extensive endo-/phago-/lysosomal system (Sections -4.2 and 6). Clearly, ciliates have increased their SNARE repertoire independently of, but in parallel to the evolution of multicellular organization.

3.2. Role of the SNARE-specific chaperone, NSF

NSF is a hexameric AAA-type ATPase with characteristic domain structure (Hanson and \Vhiteheart, 2005; \Vhiteheart et aL, 20(1). NSF is generally believed to be engaged in disentangling SNARE complexes after fusion, so SNAREs can become amenable to reuse (Littleton et al., 2001). Another possibility, though less considered in the literature, is the establishment of SNARE complexes during membrane-to-membrane attachment (Ungermann and L:mgosch, 2005; own data in Sechon 3.2.2).

To appreciate the significance of NSF one has to bear in mind the following details. Fusion capacity in /Ji/Jo depends on the assembly of a quarternary complex of a-helices from the SNARE domains. This includes a v (R)-SNARE and two or three t (Q)-SNAREs (Sections 2.1, 3.1, and 5), among them Qa-, Qb-, Qc-, and Qb/c-SNAREs (Fasshauer et aL, 1998).

Normally, a Qbl c with two pin-shaped (antiparallel) a-helical SNARE dOlnains or, more rarely, separate Qb and Qc SNAREs are superimposed to a minimal SNARE pin of tVIO membrane-anchored SNAREs (Fnkncb et al., 2(00). Thus, the minimum required for fusion is an R- and a Qa-SNARE (Gmte et al, 200Cf; l\kNe,v et at, 2(00). Specifically, in vitro studies have demonstrated that one type ofv-/R-SNARE and one type of t-/Q-SNARE suffice to mediate fusion as long as they are inserted (by their carboxy terminus) in opposing membranes, so they can form "SNARE pins" (Fasshauer et al., 1998; Graf et al, 2()05;lvlalsam et al., 2008; 1'v1eIia et al., 2()02; Sore-men et al, 20(6). Beyond the :filet that in /Ji/Jo one SNARE complex is Illade of one v-/R- and three SNARE domains from two or three t-/Q-SNAREs (Section 2.Ll) (Fukuda et al., 2000; Jahll and Scheller, 2006; 1'vlalsam et al., 20(8) it then has been found, moreover, that several such complexes are radially arranged around a potential fusion site (Section 5). All this arrangement depends on the SNARE-specific chaperone, NSF. NSF starts binding after preceding binding of !X-SNAP to the Habc domain of synt3..'(in (Bock and Scheller, 1996; Rizo and 5":11 ^f' 2Ce') X . ] 'J(09) (5" ' ')11'

cue 10', J )~; ^J. U et a., .'17 ^c ectlOn "-< • • ) .