We characterized two new CPX isoforms, CPXs III and IV, which form a novel mammalian subfamily of the CPX superfamily of SNARE regulators. A protein sequence comparison between CPXs from different species revealed only limited evolutionary conservation restricted to the region responsible for SNARE complex binding (residues 48–70 of CPX I; A). In this region, residues that based on structural data (Chen et al., 2002
) are thought to be essential for the interaction with the SNARE complex, and which we found to be essential for SNARE complex binding in mutagenesis studies (unpublished data), are conserved among isoforms and species (R48
, and Y70
of CPX I). Outside the SNARE complex binding region, evolutionary conservation is limited to certain species or one of the two CPX subfamilies. The accessory α-helix that flanks the SNARE complex binding region NH2
-terminally (residues 30–47 in CPX I) is conserved among vertebrate CPXs I and II and also insect CPXs I, but not in CPXs I from other species or in the CPX III/IV subfamily. In the latter, it is interrupted by a short, partially conserved sequence stretch. Within the NH2
termini of CPXs, only one residue (F3
in CPX I) is conserved among all isoforms and species. In contrast, a cluster of positive residues COOH-terminal of the SNARE binding region is conserved in all members of the CPX I/II subfamily, irrespective of the species, whereas it is absent from the vertebrate CPX III/IV subfamily. The most striking difference between the vertebrate CPX subfamilies is apparent at their COOH terminus where CPXs III and IV carry an extension with a functional CAAX-box farnesylation site that is absent in CPXs I and II. This CAAX-box is not a recent evolutionary acquisition because homologous sequences, in some cases also at the end of COOH-terminal extensions, are present in many invertebrate CPXs ( A). Apart from the CAAX-box, most of the invertebrate CPXs are more similar to CPXs I and II than to CPXs III and IV ( B). These data indicate that the vertebrate CPXs originate from a common ancestor with a COOH-terminal extension and a CAAX-box, both of which were lost in vertebrate members of the CPX I/II subfamily after separation of the CPX I/II and CPX III/IV subfamilies.
Our expression studies in hippocampal neurons demonstrate that farnesylation of CPXs III and IV at the CAAX-motif mediates their specific presynaptic targeting (). Given the evolutionary conservation of CAAX-motifs in CPXs (CAAM in mouse and human CPXs III and IV, CAAS in Ciona intestinalis
, and CAAQ in Hirudo medicinalis
, Drosophila melanogaster
, Anopheles gambia
, and Loligo pealeii
, all of which can be farnesylated; Zhang and Casey, 1996
; A), the same is likely to be true for all other CPXs carrying this motif. The underlying mechanism that causes synaptic targeting of prenylated CPXs remains to be elucidated. It is possible that after synthesis, lipidic or proteinaceous interactions at the level of the trans-Golgi network recruit prenylated CPXs into distinct presynapse-bound membrane compartments with characteristic lipid and protein composition.
The main function of CPXs I and II in SNARE mediated synaptic exocytosis is thought to involve stabilization of SNARE complexes in a tight conformation, thus maintaining a highly Ca2+
sensitive pool of readily releasable vesicles (Reim et al., 2001
; Chen et al., 2002
). Our data indicate that the novel CPX isoforms III and IV act as positive regulators of neurotransmitter release, as do CPXs I and II in the central nervous system, because CPXs III and IV bind to SNARE complexes ( A) and their overexpression rescues the reduced Pvr
phenotype of CPX I/II DKO neurons (, B–E). Interestingly, farnesylation of CPX III has little impact on its function under conditions of Semliki Forest Virus mediated overexpression whereas farnesylation of CPX IV is essential for its rescue function. It is likely that in the autaptic culture, the effect of loss of CPX III farnesylation can be overcome by its high binding affinity to the SNARE complex ( A). However, the binding affinity of CPX IV to the SNARE complex is much lower than that of CPXs I, II, and III ( A). As a consequence, its membrane association by farnesylation is a critical parameter to reach sufficient local CPX IV concentration for functional SNARE complex binding. This is demonstrated by the almost complete lack of rescue activity of the farnesylation-deficient CPX IV-C158S mutant (, B–E), and represents a unique feature of the ribbon synapse specific CPX IV and its role in controlling neurotransmitter release as compared with other CPX family members.
Our data indicate that prenylation and synaptic targeting of CPXs III and IV are of significant functional relevance. This implies that the loss of the CAAX-box motif in CPXs I and II must be compensated during evolution by other changes in CPX I/II protein structure or the regulation of CPX I/II protein expression in the many neurons of the central nervous system that only express CPXs I and II. Indeed, CPXs I and II appear to bind SNARE complexes with higher affinity than CPX IV ( A). Our Northern blot data ( A) as well as the relative frequency of entries in current EST databases indicate that CPXs I and II are expressed at much higher levels in brain than CPX III. Both of these characteristics, i.e., higher expression levels in brain and high SNARE complex affinity, which may be caused by the unique structural features of CPXs I and II (e.g., in the region of the accessory α-helix and the positively charged cluster COOH-terminal to the SNARE binding region), could in principle contribute to a compensation of the disadvantage that is caused by the loss of COOH-terminal farnesylation, consequent lack of synaptic targeting, and free diffusibility in neurons and their synaptic terminals.
COOH-terminal prenylation of CPXs is unlikely to interfere significantly with SNARE complex binding in vivo because CPXs associate with the SNARE complex in an antiparallel fashion (Chen et al., 2002
) such that the prenylated COOH terminus lies at the distal tip of the assembled trans SNARE complex and not at the site of vesicle and plasma membrane contact where v- and t-SNARE proteins are inserted into membranes. A systematic mutagenesis study performed in our laboratory indicates that only mutations of conserved CPX I residues in the helical region that contacts the SNARE complex interfere with SNARE complex binding. In contrast, mutations in the NH2
- and COOH-terminal sequences flanking the SNARE binding region do not interfere with SNARE complex binding of CPX I (unpublished data). Thus, structural changes in sequences outside of the actual SNARE complex binding surface of CPXs do not interfere with SNARE complex binding, and the same would be expected for the COOH-terminal farnesylation. That this is indeed the case is supported by our functional rescue experiments on CPX I/II double-deficient neurons.
Our electrophysiological rescue experiments in neurons show that the novel CPX isoforms act as positive regulators of synaptic exocytosis, as do CPXs I and II, and that farnesylation of CPX IV increases its activity. Thus, in addition to protein expression levels, the efficacy of CPX action is defined by two functional parameters, SNARE complex affinity and local membrane concentration at the site of vesicle fusion, with the latter parameter being positively modulated by COOH-terminal farnesylation. The case of CPX IV exemplifies that these two parameters can influence each other in a compensatory manner as wt CPX IV (low SNARE complex affinity but high local concentration at synaptic membranes due to farnesylation) is functionally equivalent to CPX I (high SNARE complex affinity but no local enrichment due to the lack of farnesylation), whereas the farnesylation-deficient CPX IV-C158S mutant is functionally compromised (low SNARE complex affinity and no local enrichment due to the lack of farnesylation; , B–E). In view of these considerations, CPX III, in which high SNARE complex affinity and COOH-terminal farnesylation are combined, may represent a particularly effective CPX. Thus, in terms of CPX function, ribbon synapses in the retina have all possible advantages, i.e., the high CPXs III and IV expression levels that are otherwise only found for CPXs I and II at conventional synapses of the central nervous system, and the added value of specific synaptic targeting and plasma membrane anchoring.
CPX III is present at ribbon and conventional synapses in the retina, whereas CPX IV seems to be solely expressed at ribbon synapses. The main CPX form at cone ribbon synapses is CPX III, and at rod ribbon synapses CPX IV. Rods and cones exhibit two kinetically distinct phases of release, a slow component, which is similar in both cell types, and a fast component, which is 10 times faster in cones than in rods (Rabl et al., 2005
). With CPXs III and IV we have found constituents of the presynaptic exocytotic machinery that are differentially expressed between rod and cone ribbon synapses. It is possible that the high affinity SNARE-binding, farnesylated CPX III isoform is responsible for the very fast release component in cone photoreceptors.
We postulate that CPXs III and IV contribute to the unique efficacy of transmitter release at retinal ribbon synapses. Their expression and localization in retinal ribbon synapses and their structure and targeting to synapses are characteristics of CPXs III and IV that set them apart from the CPXs present in conventional synapses. These features are also likely to convey particularly efficient release properties to the brain synapses that express CPX III.