Targeting to the plane of cell division
Many plasma membrane-localised proteins are known to accumulate at the plane of cell division during cytokinesis, suggesting that trafficking to the division plane is mechanistically related to trafficking to the plasma membrane in interphase (Reichardt et al., 2007). This includes cell wall-modifying enzymes such as endoxyloglucan transferase that are secreted from the cell in interphase and are targeted to the cell plate in dividing cells (Yokoyama and Nishitani, 2001). This has led to the notion that trafficking to the plasma membrane or the plane of cell division occurs by default (Jürgens and Pacher, 2003). There is good evidence for the idea of a default pathway to the plasma membrane, since a secretory form of soluble GFP, which is translocated across the ER membrane because of its N-terminal signal peptide, is secreted from the cell (Batoko et al., 2000). However, a fairly large number of plasma membrane-localised proteins are endocytosed during cytokinesis and then targeted from endosomes to the plane of cell division rather than being recycled to the plasma membrane (Reichardt et al., 2007). There are a few known exceptions to this rule, KNOLLE being one of them. KNOLLE is specifically synthesised during M-phase, targeted to the plane of cell division and degraded in the vacuole at the end of cytokinesis (Völker et al., 2001; Reichardt et al., 2007). Our analysis of KNOLLE-PEP12 chimeras revealed that the C-terminal membrane anchor (tail anchor) is not critical for sorting between the plane of cell division and the PVC. This is unlike the situation of proteins with a transmembrane domain, which traffic to the plasma membrane only if their transmembrane domain exceeds a certain length (Brandizzi et al., 2002). In contrast, KNOLLE-PEP12 chimeric proteins were targeted to the plane of cell division if the N-terminal region including helix Ha and the linker to helix Hb were from KNOLLE but trafficked to the PVC or vacuole if this critical region was from PEP12, regardless of the length of the swapped sequence and the origin of the remainder of the chimera. The simplest interpretation of these results would be that the sorting signal resides there and might promote vacuolar trafficking and/or prevent trafficking to the cell plate. Alternatively, KNOLLE might have multiple sorting signals in different regions of the protein but the vacuolar sorting signal of PEP12, which resides in the N-terminal region, is epistatic to them all. This rather unlikely interpretation is formally equivalent to the much simpler assumption that PEP12 has a positive sorting signal whereas KNOLLE has none. Experimentally, the two interpretations cannot be distinguished. For example, both interpretations would be compatible with the observed trafficking to the cell plate of KNOLLE proteins that have small N-terminal subfragments replaced with those of PEP12.
Deleting this critical region still resulted in the accumulation of KNOLLE (transgene #17) at the plane of cell division, whereas PEP12 (transgene #18) trafficking to the PVC/MVB appeared compromised. Although some truncated PEP12 was localised in a pattern akin to that of full-length PEP12, others were seemingly retained along the pathway, including possible retention in the ER. That some truncated PEP12 appears to localise like full-length PEP12 was unexpected, considering the observation that KNOLLE-PEP12 (transgene #5) reaches the plane of cell division, which suggests that there are no positive sorting signals to the vacuole in truncated PEP12 or KNOLLE sorting to the division plane is epistatic to those vacuolar signals. However, the reciprocal chimera PEP12-KNOLLE (transgene #4) takes the vacuolar route, suggesting the opposite epistatic relationship, if any. Another possible explanation for the vacuolar trafficking of some truncated PEP12 might be that such protein might tend to fold abnormally and thus be subject to vacuolar degradation.
The significance of this reduced trafficking efficiency of PEP12 is not clear at present. Conceivably, sorting of PEP12 might already occur at the exit from the ER whereas KNOLLE seems to ‘go with the flow’. Alternatively, KNOLLE and PEP12 might use different ER exit signals that are located in different regions of the two syntaxins but are both recognised by the COPII subunit SEC24. For example, a di-acidic peptide consisting of five amino acids (MELAD) residing in the linker between Hb and Hc appears to mediate export of Golgi-localised SYP31 from the ER (Chatre et al., 2009). In yeast, however, Sec24p has distinct binding sites for the SYP31 orthologue Sed5p and other SNAREs, recognising a different signal residing in the linker between Hc and the SNARE domain of Sed5p (Miller et al., 2003, 2005). Interestingly, chimeras with the N-terminal region of mixed KNOLLE–PEP12 origin were largely retained at the Golgi/TGN (if not trafficked to the plane of cell division), suggesting that sorting to the vacuolar pathway was incomplete or defective. All these observations support the notion that syntaxin trafficking to the plane of cell division occurs by default, i.e. if there is no signal that mediates sorting to some alternative endomembrane destination, as analysed here for PEP12. How default traffic to the plane of cell division is brought about mechanistically is not clear at present. Because both newly synthesised and endocytosed membrane proteins accumulate there, it seems reasonable to presume that the phragmoplast microtubules provide some guidance to the targeting process. Indeed, KNOLLE is dispersed in the dividing cell rather than accumulating at the plane of division when the formation of microtubules is blocked in mutant embryos lacking tubulin-folding cofactors (Mayer et al., 1999). However, no kinesin motor protein has been identified that would move the cytokinetic vesicles to the plane of cell division.
In general, not much is known about the targeting of syntaxins to their site of action in any system (Salaün et al., 2004). Interestingly, the plasma membrane-localised syntaxin 3 is prevented by its SNARE domain from entering the outer segment area of photoreceptor cells, which appears to be the default targeting area of the plasma membrane (Baker et al., 2008). In epithelial cells, syntaxin 3 is targeted to the apical domain of the plasma membrane, which appears to require a four amino-acid motif, FMDE, in helix Ha but not the SNARE domain (Sharma et al., 2006). Syntaxin 4 is targeted to the complementary baso-lateral area of the plasma membrane of epithelial cells, which appears to require a small motif N-terminal to helix Ha (Torres et al., 2011). Thus, different parts of syntaxin mediate sorting in positive or negative ways, and there seems to be no unifying principle.
Which parts of KNOLLE are required for action of syntaxin at the division plane?
When KNOLLE-related SYP1 syntaxins were tested for their ability to substitute for KNOLLE when expressed from the KNOLLE promoter, the most closely related syntaxin SYP112 rescued the knolle mutant whereas PEN1 (SYP121) was unable to do so (Müller et al., 2003). Here, we swapped domains between the SYP1 syntaxin KNOLLE and the SYP2 syntaxin PEP12 in order to identify relevant features for syntaxin function in cytokinesis. Interestingly, both the very N-terminus and the very C-terminus of KNOLLE could be exchanged with those of PEP12 without compromising the knolle-rescuing ability of the chimeric protein. This is interesting because other syntaxins have been shown to require a specific N-peptide motif for interacting with cognate SM protein (Südhof and Rothman, 2009). In contrast, deletion of the N-terminal region up to the start of helix Hb rendered the truncated KNOLLE protein unable to rescue the knolle mutant, although trafficking to the plane of cell division was not affected. Thus, the N-terminus might contain a sequence-unspecific element essential for syntaxin action at the plane of cell division. However, we cannot exclude the possibility that N-terminally truncated KNOLLE protein might be structurally altered in such a way that its activity is impaired without compromising its trafficking. Furthermore, a major part of the N-terminal helical region, including the linker preceding helix Hb, both helices Hb and Hc and the interjacent linker when derived from PEP12, did not interfere with syntaxin function of the chimeric protein in cytokinesis. In contrast, the SNARE domain and the linker preceding this domain had to be of KNOLLE origin for the chimeric syntaxin to rescue the knolle mutant. The sequence-specific requirement for the SNARE domain from KNOLLE can be easily related to its interaction with cognate partners to form SNARE complexes that mediate membrane fusion (McNew et al., 2000). Very recently, PEN1 (SYP121) was relieved of its inability to rescue the knolle mutant by replacing its SNARE domain with that of KNOLLE (Reichardt et al., 2011). In contrast, the requirement for the linker domain was unexpected and its role in syntaxin function during cytokinesis is at present unknown. It remains to be determined whether the sequence-specific requirement of the linker is unique to KNOLLE and what its functional significance might be.