In eukaryotic cells, the trafficking of vesicular compartments to specific subcellular locations is mediated by distinct families of proteins with evolutionarily conserved functions (). At the heart of this machinery are the ‘SNARE’ proteins (soluble N
-ethylmaleimide–sensitive factor accessory protein receptors), which are helical transmembrane proteins found in either target membranes (t-SNARE proteins) or vesicles (v-SNARE proteins)27
. In the appropriate circumstances, v-SNARE and t-SNARE proteins form hetero-oligomers of coiled coils that promote vesicle fusion. Different v-SNARE and t-SNARE proteins localize to distinct subcellular compartments, and there is evidence that they interact with a fair amount of specificity28
. SNARE interactions are regulated by a diverse group of accessory proteins, which impart an additional layer of specificity to the trafficking process. In neurons, for example, the SNARE-binding protein Munc13-1 facilitates the interaction of v-SNARE and t-SNARE proteins at the pre-synaptic membrane29
. Vesicle fusion in neuronal synapses is also regulated by synaptotagmin, which couples calcium influx to the formation of SNARE complexes30
. Particularly important for the specification of intracellular traffic is the Rab family of small GTPases31
. Rab proteins associate with many vesicular structures by virtue of post-translational prenylation at their carboxyl termini. They function by recruiting various effector molecules and tethering complexes that specify the identity, and hence the trafficking activity, of the underlying vesicle. Over 60 Rab proteins are expressed in higher mammals, and each defines a distinct pool of vesicles.
Figure 2 Secretion of vesicles from cells. The secretion of synaptic vesicles at the neuronal synapse (left) is compared with what is known about the secretion of lytic granules (center) and synaptic and multidirectional cytokines (right) from activated T cells. (more ...)
Studies have identified several trafficking proteins important for the release of lytic granules from CTLs (). A particularly productive approach has been the analysis of genetic disorders that combine immunodeficiency with albinism. Both CTLs and melanocytes secrete secretory lysosomes (lytic granules and melanosomes, respectively), and at least some of the trafficking machinery is probably conserved. Griscelli syndrome, which is characterized by albinism and defects in the cytotoxicity of CTLs and natural killer cells, is caused by the functional loss of Rab27a32
. In T cells derived from Rab27a-deficient mice, lytic granules polarize toward the IS in response to TCR stimulation but fail to dock33
. Consistent with involvement in docking, the amount of Rab27a associated with lytic granules seems to increase as they approach the IS. Notably, in CTLs missing the geranylgeranyl transferase required for Rab prenylation, lytic granules fail to polarize, which suggests that other Rab proteins are involved in earlier phases of granule trafficking33
. Hermansky-Pudlak syndrome type 2 also combines immunodeficiency with albinism and results from mutations in the gene encoding adaptor protein 3 (AP-3)34
. AP-3 has been linked to the sorting of lysosomal cargo in many cell types35
. AP-3-deficient CTLs have enlarged lytic granules that do not polarize to the IS, which suggests involvement of AP-3 in directional trafficking34
. Of note, a similar defect in granule polarization has been reported in CTLs lacking protein kinase C-δ36
and also in natural killer cells deficient in the cytoskeletal adaptor protein WIP37
. Whether protein kinase C-δ and WIP work in concert with AP-3 or are involved in a distinct step during translocation remains to be seen.
Familial hemophagocytic lymphohistiocytosis (FHL) represents another set of genetic disorders associated with secretory defects in CTLs but not melanocytes38
. Killer lymphocytes from patients with FHL are deficient in their ability to deliver perforin to the IS. FHL type 2 is caused by a loss of perforin itself38
. FHL type 3, in contrast, results from mutations in the gene encoding Munc13-4, a homolog of Munc13-1 (ref. 39
). In CTLs derived from patients with FHL type 3, lytic granules dock at the IS but fail to fuse. Munc 13-4 interacts with Rab27a and localizes to lytic granules39,40
. These data collectively suggest that Munc13-4 acts ‘down-stream’ of Rab27a in the granule secretion process. FHL type 4 (FHL4), which is clinically similar to but less severe than FHL type 3, is caused by defects in syntaxin-11 (ref. 41
), a SNARE protein. This deficiency in syntaxin-11 impairs granule exocytosis without affecting polarization, which suggests an important function ‘downstream’ of the recruitment of granules to the IS.
The functional relevance of each of those proteins for cytolysis is now well established. Determining how they contribute mechanistically to the trafficking and secretion of lytic granules will be the next important challenge. It can be speculated that proteins such as Rab27a and Munc13-4 function at positions in the secretory pathway analogous to those of their structural homologs in neurons (). However, it remains to be determined whether this is indeed the case. In vitro
systems able to distinguish the many component steps of granule exocytosis will be needed to resolve this issue. The identification of interacting proteins and additional required molecules will also be instrumental. Of note, a study has found that Rab27a interacts with the synaptotagmin-like proteins Slp1 and Slp2, which could both be involved in the docking of granules at the IS42
. Another important area of investigation is the regulation of granule trafficking by intracellular signaling pathways. In T cells, it is known that both calcium signaling and activity of the kinase Erk are required for efficient degranulation43,44
. The calcium-regulated proteins required for the secretion of lytic granules have not been defined. For the most part, the relevant Erk substrates also remain elusive. A good candidate is paxillin, a cytoskeletal scaffolding protein that has been studied as a component of focal adhesions45
. Paxillin is associated with the MTOC in resting T cells, but after antigen recognition, it undergoes Erk-dependent phosphorylation and transits to the IS43
. Further studies will be needed to determine the functional importance of this activity.