Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4⌬608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion. INTRODUCTIONRab GTPases serve as important regulators of membrane transport in eukaryotic cells (Deneka et al., 2003b). Most rabs are ubiquitously expressed, but some have a more restricted distribution. For instance, rab27a is highly expressed in melanocytes and hematopoietic and other secretory cells (Tolmachova et al., 2004). Mutations causing loss of rab27a function in human result in defects of pigmentation (Bahadoran et al., 2001) and defects in the granule exocytosis pathway in cytotoxic T lymphocytes (CTLs) (Menasche et al., 2000). This rare autosomal recessive disease is called Griscelli syndrome type II (GS2) (Sanal et al., 2002).Mutations in the genes encoding myosin-Va and the rab27a effector melanophilin also cause a pigmentation phenotype in human (Pastural et al., 1997;Menasche et al., 2003) and mice. These observations led to the identification of a ternary complex consisting of rab27a/melanophilin/myosin-Va that is essential for the normal function of melanosomes (Wu et al., 2002). A related complex containing rab27a/myRIP/myoVIIa seems to be important for melanosome localization in retinal pigment epithelium (Amraoui et al., 2002) and secretory granules in the PC12 pheochromocytoma cell line (Desnos et al., 2003).Melanocytes and hematopoietic cells combine the functions of lysosomes and secretory granules into a hybrid organelle, the melanosome and secretory lysosome, respectively. Secretory lysosomes are particularly found in cells of the hematopoietic lineage, such as natural killer cells, CTLs, mast cells, dendritic cells, B cells, and neutrophils. They have an acidic lumenal pH and contain lysosomal enzymes. Ne...

Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4-rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4-rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5a and its putative partner g 1 -adaptin in membrane recycling. We found that rabaptin-5a forms a ternary complex with the g 1 ±s 1 subcomplex of AP-1, via a direct interaction with the g 1 -subunit. The binding site for g 1 -adaptin is in the hinge region of rabaptin-5a, which is distinct from rab4-and rab5-binding domains. Endogenous or ectopically expressed g 1 -adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5a and g 1 -adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5a caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5a± g 1 -adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5a and g 1 -adaptin regulate membrane recycling.

Membrane flow through the cell is a highly dynamic process in which intracellular compartments communicate via tubulo-vesicular structures shuttling cargo molecules to their destinations. Transport carriers are formed at a donor compartment and navigate through the cytoplasm to the target organelle, on which they subsequently dock and fuse. Many of these events are regulated by the cooperative action of monomeric rab GTPases and their effector proteins. Research in recent years resulted in the identification of many rab effectors, providing first glimpses how the GTPase switch of individual rab proteins is utilized in discrete transport steps.