A TOGL domain specifically targets yeast CLASP to kinetochores to stabilize kinetochore microtubules.

Cytoplasmic linker-associated proteins (CLASPs) are proposed to function in cell division based on their ability to bind tubulin via arrayed tumor overexpressed gene (TOG)-like (TOGL) domains. Structure predictions suggest that CLASPs have at least two TOGL domains. We show that only TOGL2 of Saccharomyces cerevisiae CLASP Stu1 binds to tubulin and is required for polymerization of spindle microtubules (MTs) in vivo. In contrast, TOGL1 recruits Stu1 to kinetochores (KTs), where it is essential for the stability and tension-dependent regulation of KT MTs. Stu1 is also recruited to spindle MTs by different mechanisms depending on the mitotic phase: in metaphase, Stu1 binds directly to the MT lattice, whereas in anaphase, it is localized indirectly to the spindle midzone. In both phases, the activity of TOGL2 is essential for interpolar MT stability, whereas TOGL1 is not involved. Thus, the two TOGL domains of yeast CLASP have different activities and execute distinct mitotic functions.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion.

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p.

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.

The cancer antigen CA125 represents a novel counter receptor for galectin-1.

CA125 is an ovarian cancer antigen whose recently elucidated primary structure suggests that CA125 is a giant mucin-like glycoprotein present on the cell surface of tumor cells. Here, we establish a functional link between CA125 and beta-galactoside-binding, cell-surface lectins, which are components of the extracellular matrix implicated in the regulation of cell adhesion, apoptosis, cell proliferation and tumor progression. On the basis of mass spectrometry and immunological analyses, we find that CA125 is a counter receptor for galectin-1, as both soluble and membrane-associated fragments of CA125 derived from HeLa cell lysates are shown to bind specifically to human galectin-1 with high efficiency. This interaction is demonstrated (1) to depend on beta-galactose-terminated, O-linked oligosaccharide chains of CA125, (2) to be preferential for galectin-1 versus galectin-3 and (3) to be regulated by the cellular background in which CA125 is expressed. Despite lacking a conventional signal peptide, a CA125 C-terminal fragment of 1148 amino acids, representing less than 10% of the full-length protein, retains the ability to integrate into secretory membranes such as the endoplasmic reticulum (ER) and the Golgi, and is targeted to the plasma membrane by conventional secretory transport. As demonstrated by a novel assay that reconstitutes non-conventional secretion of galectin-1 based on fluorescence-activated cell sorting (FACS), we find that tumor-derived HeLa cells expressing endogenous CA125 present more than ten times as much galectin-1 on their surface compared with non-tumor-derived, CA125-deficient CHO cells. Intriguingly, both the galectin-1 expression level and the cell-surface binding capacity for galectin-1 are shown to be similar in CHO and HeLa cells, suggesting that CA125 might be a factor involved in the regulation of galectin-1 export to the cell surface.

Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation.

We show here that Ask1p, Dad2p, Spc19p and Spc34p are subunits of the budding yeast Duo1p-Dam1p- Dad1p complex, which associate with kinetochores and localize along metaphase and anaphase spindles. Analysis of spc34-3 cells revealed three novel functions of the Duo1-Dam1p-Dad1p subunit Spc34p. First, SPC34 is required to establish biorientation of sister kinetochores. Secondly, SPC34 is essential to maintain biorientation. Thirdly, SPC34 is necessary to maintain an anaphase spindle independently of chromosome segregation. Moreover, we show that in spc34-3 cells, sister centromeres preferentially associate with the pre-existing, old spindle pole body (SPB). A similar preferential attachment of sister centromeres to the old SPB occurs in cells depleted of the cohesin Scc1p, a protein with a known role in facilitating biorientation. Thus, the two SPBs are not equally active in early S phase. We suggest that not only in spc34-3 and Deltascc1 cells but also in wild-type cells, sister centromeres bind after replication preferentially to microtubules organized by the old SPB. Monopolar attached sister centromeres are resolved to bipolar attachment in wild-type cells but persist in spc34-3 cells.