Research SummaryRole of Hermansky-Pudlak Syndrome associated protein complexes in organelle biogenesis
Biochemical reactions within eukaryotic cells are compartmentalized by intracellular membranes into distinct membrane-bound organelles. Each organelle is different and conducts sets of competing or incompatible reactions. Cells achieve this process by sorting macromolecules - including proteins and delivering them to proper place, which involve intracellular sorting mechanisms. My laboratory is interested in understanding how this process works.
Key players in transport pathways for melanin synthesis
Delivery of cargo to the correct address requires accurate directions and dependable machinery for delivery. Any defects in them affect timely delivery or cause the cargo to be misdelivered. This chain of events is not very different at the cellular level. Cells have their own transport pathways responsible for cargo delivery to right destination and timely manner (Jani and Setty, 2014, Resonance). Any deficiencies in these pathways show up as mild symptoms, or may cause fatal diseases. We are interested in understanding one such transport pathway in specialized animal cells where failure to deliver the cargo such as melanin synthesizing enzymes could result in disorders like albinism.
Melanin pigments are responsible for the colour of our skin and also play an important role in protection against radiations and other damages from light. They are produced by a series of organic chemical reactions that occur in cellular organelles called melanosomes, a type of lysosome-related organelle (LRO), coexist with other cellular organelles including lysosomes. Melanosome biogenesis occurs in four different stages (I to IV) and this process requires primary melanin-synthesizing enzymes such as tyrosinase (TYR, initiates pigmentation), tyrosinase-related protein (TYRP1, functions down-stream in the melanin biosynthesis pathway) and other enzymes including structural protein like pre-melanosomal protein (PMEL, forms amyloid fibers). In addition, PMEL initiates the fiber formation in late-endosomes and matures into pre-melanosomes (stage II). Later, the proteins TYR and TYRP1 are transported from recycling endosomes to stage II by two independent transport mechanisms that generate the stage III and IV mature melanosomes. Moreover, we and others have shown that these cargo transport steps are facilitated by four multi-subunit cytosolic protein complexes, BLOC (biogenesis of lysosome-related organelles complex)-1, -2, -3 and adaptor protein (AP)-3. However, the precise function of these complexes is only partly known.
BLOC-1 consists of eight subunits, functioning in the transport of TYRP1 (BLOC-1-dependent cargo) and AP-3, a tetra-subunit complex mediate the transport of TYR (AP-3-dependent cargo) to maturing melanosomes. However, the function of BLOC-2, a three-subunit protein complex, in regulating cargo transport or biogenesis pathways is unknown. We have elucidated recently that the role of BLOC-2 in directing the cargo containing endosomal tubules/vesicles towards maturing melanosomes for subsequent delivery. Our studies using live imaging and electron microscopy suggest that BLOC-2 regulates this step either by tethering the cargo containing tubules with maturing melanosomes during membrane fusion or stabilizing the cargo containing tubular structures during the delivery (Dennis et al., 2015, J. Cell Biol.). Moreover, we have shown that mutations in any protein subunit of BLOC-2 result in inefficient delivery of melanin synthesizing proteins to maturing melanosomes besides mistargeting the cargo to other organelles (Golgi, plasma membrane, endosomes and lysosomes) and thus failure in production of the melanin pigment/organelle biogenesis. This malfunction manifests in the form of albinism of skin and eye, also referred to as oculocutaneous albinism. In addition, this phenotype along with other symptoms such as lung fibrosis and bleeding is commonly observed in Hermansky-Pudlak Syndrome (HPS) patients. Out of the 16 possible genetic mutations that can result in HPS, so far only 9 are known in humans. Three out of these nine subtypes are a result of mutations in the BLOC-2 protein complex (Dennis et al., 2015, J. Cell Biol.).
Even though it is now established that BLOC-1, -2 and AP-3 play key roles in the overall cargo delivery, the mechanism of how they achieve this is not yet clear. In addition to these key proteins, other cellular machineries are also known to be responsible for membrane fusion and cargo delivery. These proteins are called Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein REceptor (SNARE). SNARE proteins, a family of about 38 proteins has been known for their role in membrane fusion during the delivery of cargo. For the first time, we have identified two members from the SNARE family such as STX13 (recycling endosomal SNARE) and VAMP7 (melanosome bound SNARE) that are involved in the protein transport pathways to melanosomes. Loss in expression of these proteins mistargets and degrades the enzymes/cargo, and affects melanosome maturation. Moreover, we have demonstrated that higher expression of a mutant (N-terminal regulatory domain deleted) form of STX13 increased cell pigmentation possibly by raising the SNARE/membrane fusion activity. Furthermore, the mutant is mislocalized to the melanosomes suggesting that regulatory domain of the STX13 controls the SNARE activity and is also required for its recycling from melanosomes to recycling endosomes. Additionally, our study has also shown how STX13 cross talks with VAMP7 for efficient cargo delivery and melanosome formation (Jani et al., 2015, J. Cell Sci.). Taken together, these studies show that BLOC-2 directs the cargo containing recycling tubular endosomes to maturing melanosomes, and STX13-VAMP7 SNAREs together mediate the membrane fusion between these organelles.
Studies from other groups have shown that BLOC-3, a two-subunit protein complex functions in recruiting small GTPases such as Rab38/32 on to the melanosome membranes and regulate the cargo delivery to melanosomes. However, it is not clear how the intramolecular interactions occur between the SNAREs, BLOC complexes, Rabs and other key players involved in the transport pathway. Recently, we have demonstrated that Rab9A (another GTPase) plays a role in regulating cargo delivery to maturing melanosomes. Loss in expression of Rab9A mistargets the melanosome cargo (TYR and TYRP1) to lysosomes and inhibits the pigment granule biogenesis. Moreover, we have shown that a cohort of Rab9A localizes to melanosomes and functions similar to and possibly upstream of BLOC-3, Rab38/32 and VARP (a VAMP7 binding protein) molecules. Further, our studies have illustrated that Rab9A and its downstream proteins very likely control the delivery of cargo containing STX13-positive tubular structures towards the melanosomes for its maturation (Mahanty et al., 2016, Pigment Cell Melanoma Res.). Additionally, we have implicated that this process is possibly conserved in the formation of other LROs such as dense granules in platelets and lamellar bodies in type II lung epithelial cells, which are also defective in some HPS patients and their biogenesis process is poorly understood (Jani et al., 2016, Bioarchitecture). In future, we would like to study the mechanism of recruitment of BLOCs, Rabs and other key molecules onto the membranes, which would help us in understanding the biogenesis of specialized organelles. Nonetheless, these studies will form the basis for organelle biogenesis in addition to understanding the etiology of an autosomal recessive disorder.
Figure Legend: Bright-field and immunofluorescence microscopy image of wild-type mouse melanocytes expressing mutated STX13 (a recycling endosomal Qa-SNARE), which results in its mislocalisation to melanosomes. Green, mutant STX13; red, a melanosome-resident protein TYRP1; blue, pseudocoloured melanosomes captured with bright-field microscopy. See article by R. A. Jani et al (2015).