Santiago M. Di Pietro

The Cell Biology of Hermansky–Pudlak Syndrome: Recent Advances

Hermansky–Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding. These manifestations arise from defects in the biogenesis of lysosome‐related organelles, including melanosomes and platelet dense granules. Most genes associated with HPS in humans and rodent models of the disease encode components of multisubunit protein complexes that are expressed ubiquitously and play roles in intracellular protein trafficking and/or organelle distribution. A small GTPase of the Rab family, Rab38, is also implicated in the pathogenesis of the disease. This article reviews recent progress toward elucidating the cellular functions of these proteins.

BLOC-2, AP-3, and AP-1 Proteins Function in Concert with Rab38 and Rab32 Proteins to Mediate Protein Trafficking to Lysosome-related Organelles

Background: Lysosome-related organelles are a group of cell type-specific compartments with specialized functions, including melanosomes in melanocytes. Results: Cell type-specific Rab proteins, Rab32 and Rab38, colocalize and interact with the ubiquitous trafficking machinery in melanocytes. Conclusion: Rab32 and Rab38 cooperate with the ubiquitous trafficking machinery for melanosome biogenesis. Significance: Learning how lysosome-related organelles are built is key to understanding their biology. Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

Characterization of BLOC-2, a complex containing the Hermansky-Pudlak syndrome proteins HPS3, HPS5 and HPS6.

Hermansky–Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding due to defects in the lysosome‐related organelles, melanosomes and platelet‐dense granules, respectively. Most HPS genes, including HPS3, HPS5 and HPS6, encode ubiquitously expressed novel proteins of unknown function. Here, we report the biochemical characterization of a stable protein complex named Biogenesis of Lysosome‐related Organelles Complex‐2 (BLOC‐2), which contains the HPS3, HPS5 and HPS6 proteins as subunits. The endogenous HPS3, HPS5 and HPS6 proteins from human HeLa cells coimmunoprecipitated with each other from crude extracts as well as from fractions resulting from size‐exclusion chromatography and density gradient centrifugation. The native molecular mass of BLOC‐2 was estimated to be 340 ± 64 kDa. As inferred from the biochemical properties of the HPS6 subunit, BLOC‐2 exists in a soluble pool and associates to membranes as a peripheral membrane protein. Fibroblasts deficient in the BLOC‐2 subunits HPS3 or HPS6 displayed normal basal secretion of the lysosomal enzyme β‐hexosaminidase. Our results suggest a common biological basis underlying the pathogenesis of HPS‐3, ‐5 and ‐6 disease.

Mechanism of platelet dense granule biogenesis: study of cargo transport and function of Rab32 and Rab38 in a model system

Dense granules are important in platelet aggregation to form a hemostatic plug as evidenced by the increased bleeding time in mice and humans with dense granule deficiency. Dense granules also are targeted by antiplatelet agents because of their role in thrombus formation. Therefore, the molecular understanding of the dense granule and its biogenesis is of vital importance. In this work, we establish a human megakaryocytic cell line (MEG-01) as a model system for the study of dense granule biogenesis using a variety of cell biology and biochemical approaches. Using this model system, we determine the late endocytic origin of these organelles by colocalization of the internalized fluid phase marker dextran with both mepacrine and transmembrane dense granule proteins. By mistargeting of mutant dense granule proteins, we demonstrate that sorting signals recognized by adaptor protein-3 are necessary for normal transport to dense granules. Furthermore, we show that tissue-specific Rab32 and Rab38 are crucial for the fusion of vesicles containing dense granule cargo with the maturing organelle. This work sheds light on the biogenesis of dense granules at the molecular level and opens the possibility of using this powerful model system for the investigation of new components of the biogenesis machinery.

The Hermansky-Pudlak Syndrome 3 (Cocoa) Protein Is a Component of the Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2)*

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease affecting vesicle trafficking among lysosome-related organelles. The Hps3, Hps5, and Hps6 genes are mutated in the cocoa, ruby-eye-2, and ruby-eye mouse pigment mutants, respectively, and their human orthologs are mutated in HPS3, HPS5, and HPS6 patients. These three genes encode novel proteins of unknown function. The phenotypes of Hps5/Hps5,Hps6/Hps6 and Hps3/Hps3,Hps6/Hps6 double mutant mice mimic, in coat and eye colors, in melanosome ultrastructure, and in levels of platelet dense granule serotonin, the corresponding phenotypes of single mutants. These facts suggest that the proteins encoded by these genes act within the same pathway or protein complex in vivo to regulate vesicle trafficking. Further, the Hps5 protein is destabilized within tissues of Hps3 and Hps6 mutants, as is the Hps6 protein within tissues of Hps3 and Hps5 mutants. Also, proteins encoded by these genes co-immunoprecipitate and occur in a complex of 350 kDa as determined by sucrose gradient and gel filtration analyses. Together, these results indicate that the Hps3, Hps5, and Hps6 proteins regulate vesicle trafficking to lysosome-related organelles at the physiological level as components of the BLOC-2 (biogenesis of lysosome-related organelles complex-2) protein complex and suggest that the pathogenesis and future therapies of HPS3, HPS5, and HPS6 patients are likely to be similar. Interaction of the Hps5 and Hps6 proteins within BLOC-2 is abolished by the three-amino acid deletion in the Hps6ru mutant allele, indicating that these three amino acids are important for normal BLOC-2 complex formation.

Regulation of clathrin adaptor function in endocytosis: novel role for the SAM domain

During clathrin‐mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin‐binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile α‐motif (SAM) domain and shows a propensity to oligomerize. By co‐immunoprecipitation, Sla1p binds to clathrin and self‐associates in vivo. Mutations in the clathrin‐binding motif that abolish clathrin binding and structure‐based mutations in SHD2 that impede self‐association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin‐mediated endocytosis.

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Background: The biogenesis of melanosomes and other lysosome-related organelles requires a pair of Rab GTPases, Rab32 and Rab38. Results: Myosin Vc is a novel binding partner of these Rabs. Myosin Vc functions in the trafficking of integral membrane proteins to melanosomes. Conclusion: Myosin Vc works in transport to and secretion of melanosomes. Significance: These results advance understanding of melanosome biology. Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.

Cell type-specific Rab32 and Rab38 cooperate with the ubiquitous lysosome biogenesis machinery to synthesize specialized lysosome-related organelles

Lysosome-related organelles (LROs) exist in specialized cells to serve specific functions and typically co-exist with conventional lysosomes. The biogenesis of LROs is known to utilize much of the common protein machinery used in the transport of integral membrane proteins to lysosomes. Consequently, an outstanding question in the field has been how specific cargoes are trafficked to LROs instead of lysosomes, particularly in cells that simultaneously produce both organelles. One LRO, the melanosome, is responsible for the production of the pigment melanin and has long been used as a model system to study the formation of specialized LROs. Importantly, melanocytes, where melanosomes are synthesized, are a cell type that also produces lysosomes and must therefore segregate traffic to each organelle. Two small GTPases, Rab32 and Rab38, are key proteins in the biogenesis of melanosomes and were recently shown to redirect the ubiquitous machinery—BLOC-2, AP-1 and AP-3—to traffic specialized cargoes to melanosomes in melanocytes. In addition, the study revealed Rab32 and Rab38 have both redundant and unique roles in the trafficking of melanin-producing enzymes and overall melanosome biogenesis. Here we review these findings, integrate them with previous knowledge on melanosome biogenesis and discuss their implications for biogenesis of other LROs.

SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

During endocytosis, actin polymerization nucleated by the Arp2/3 complex provides force needed to drive internalization. Las17 is the strongest activator of the Arp2/3 complex in yeast cells. This study shows that Las17 is associated into a stable complex with Sla1, an adaptor that inhibits Las17 activity both in vitro and in vivo.

Purification and structural characterization of a fatty acid-binding protein from the liver of the catfish Rhamdia sapo

We report here the isolation of a fatty acid-binding protein (FABP) from the liver of the catfish Rhamdia sapo. The purification procedure involves gel filtration, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The purified protein is basic (pI > 8.7) and migrates on sodium dodecyl sulfate-gel electrophoresis as a single entity of about 15 kDa. Its amino acid composition resembles those of FABPs isolated from other animals. Unlike mammalian liver FABPs, catfish liver FABP contains at least one tryptophan residue per molecule. No significant cross-reactivity was observed between the purified protein and polyclonal antibodies against either rat liver FABP or rat heart FABP. Amino acid sequencing of peptides obtained by digestion with Lys-C revealed that the catfish protein is structurally more similar to chicken liver FABP (69% identity in a 67-residue overlap) than to human liver FABPs (36%), nurse shark (Ginglymostoma cirratum) liver FABP (30%) and human heart FABP (31%). Taken together, these results suggest that catfish liver FABP is far more closely related to chicken liver FABP than to the FABPs isolated from the liver of mammals or elasmobranchs.