The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite <10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins.

Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation.

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re-expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1-phosphorylation-deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.

A cluster of amino acid residues located in the AB-GH region of the ␣-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, ␣Leu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (␤Glu-63 Val, ␣Leu-1133 His) to ascertain the hitherto unknown role of the ␣113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C sat ) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the ␣113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the ␣113 mutation was coupled individually with two established fiber contact sites (␣16 and ␣23) located in the AB region of the ␣-chain: HbS(␣Lys-163 Gln, ␣Leu-1133 His), HbS(␣Glu-233 Gln, ␣Leu-1133 His). The single mutants at ␣16/␣23 sites were also engineered as controls. The C sat values of the HbS point mutants involving sites ␣16 or ␣23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast, C sat values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of ␣113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.Sickle cell anemia is a consequence of a point mutation (Glu-63 Val) at the sixth position in the ␤-chain of the hemoglobin molecule (1). The replacement of a charged residue with a hydrophobic one on the surface of the protein drastically reduces the solubility of the deoxygenated sickle hemoglobin (HbS) 1 , leading to its polymerization into long helical fibers that are responsible for the clinical manifestations of sickle cell disease. Electron microscopy and crystallographic studies have suggested that both the deoxy HbS crystal and fiber are constructed from the same "Wishner-Love" double strands (2-5). The model of the fiber structure derived from these analyses consists of seven Wishner-Love double strands (6 -9).The polymerization process is triggered by lateral interactions of the donor Val-6␤ of a tetramer of one strand of the double strand with the acceptor pocket at the EF corner (elicited mainly by ␤Phe-85 and ␤Leu-88) of the ␤-chain of an adjacent molecule present in the second strand of the double strand. Subsequent intra-double strand and inter-double strand interactions involving several amino acid residues from both ␣-and ␤-chains contribute to the stabilization of the fiber structure. The polymerization-impairing o...

The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the alpha-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated alpha-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.