SummaryClathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show the μ-homology domain (μHD) of FCHO1/2 represents a novel endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μHD interacts with the Bmp receptor Alk8, uncovering a new endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus contradict the primacy of FCHO1/2 in coat initiation.

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.DOI: http://dx.doi.org/10.7554/eLife.04137.001

PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes.

The coding sequence for hexokinase enzyme was cloned from Leishmania major. The sequence was found to encode an enzyme with a molecular mass of 51.74 kDa. Amino acid sequence showed maximum homology with known trypanosome and plant hexokinases. It has a calculated isoelectric point of 8.46 and contains an N-terminal peroxisome-targeting signal, the characteristics frequently associated with glycosomal proteins. The sequence indicated the presence of conserved amino acid residues and motifs that are present in plant and mammalian hexokinases; these are apparently involved in the binding of different substrates. The L. major genome was found to have 2 copies of hexokinase coding sequences in tandem with an intergenic spacer of 2.58 kb. Both the genes in the hexokinase locus were transcribed as individual transcripts in a monocistronic form, having the same size as seen by Northern blot analysis. The hexokinase gene was transcribed in large amounts in the promastigote stage, whereas there is only weak expression in the amastigote stage as determined by RT-PCR analysis. Sequencing of hexokinase loci from different Leishmania species (e.g., L. donovani, L. infantum, L. tropica, and L. mexicana) revealed that the hexokinase locus is highly conserved at the DNA and protein levels among species of Leishmania compared with trypanosomes.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence–structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of L. mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.