G protein–coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The α-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding α-factor. Ubiquitination triggers Ste2p internalization into the endocytic pathway. Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization. Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis. The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence. Mutants defective in yeast casein kinase I homologues are unable to internalize α-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.

The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency. In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization. The Saccharomyces cerevisiae end8‐1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis. Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen. This block can be overcome by exogenous sphingoid base. Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect. Therefore, the required lipid is most likely a sphingoid base. Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation. This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.

Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of erg⌬ mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2⌬erg6⌬ and erg3⌬erg6⌬ cells exhibit a strong internalization defect of the ␣-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligandreceptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. erg⌬ cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment.

Coronin 1 is a member of the coronin protein family specifically expressed in leukocytes and accumulates at sites of rearrangements of the F-actin cytoskeleton. Here, we describe that coronin 1 molecules are coiled coil-mediated homotrimeric complexes, which associate with the plasma membrane and with the cytoskeleton via two distinct domains. Association with the cytoskeleton was mediated by trimerization of a stretch of positively charged residues within a linker region between the N-terminal, WD repeat-containing domain and the C-terminal coiled coil. In contrast, neither the coiled coil nor the positively charged residues within the linker domain were required for plasma membrane binding, suggesting that the N-terminal, WD repeat-containing domain mediates membrane interaction. The capacity of coronin 1 to link the leukocyte cytoskeleton to the plasma membrane may serve to integrate outside-inside signaling with modulation of the cytoskeleton. INTRODUCTIONCoronin 1 is expressed exclusively by leukocytes (Suzuki et al., 1995;Ferrari et al., 1999) and is a member of the WD repeat protein family termed coronins, which are collectively defined as F-actin-associated proteins widely expressed in the eukaryotic kingdom (de Hostos, 1999). In Dictyostelium discoideum, coronin colocalizes with F-actin filaments at crown-shaped phagocytic cups and macropinosomes (de Hostos et al., 1991(de Hostos et al., , 1993Maniak et al., 1995;Fukui et al., 1999). Dictyostelium deleted for coronin displays strong reduction in cell locomotion, phagocytosis, macropinocytosis, and cytokinesis, indicating that in this slime mold coronin is functionally involved in F-actin-based motility-related processes (de Hostos et al., 1993). In Saccharomyces cerevisiae, the single coronin isoform Crn1p was found to localize to cortical F-actin patches in an actin-dependent manner (Heil-Chapdelaine et al., 1998). In vitro, Crn1p can nucleate and cross-link F-actin filaments and bind to microtubules (Goode et al., 1999). Recently, yeast Crn1 was proposed to promote the formation of actin filament networks based on its interaction with the Arp2/3 complex (Humphries et al., 2002). Interestingly, unlike the Dictyostelium coronin-null mutant, an S. cerevisiae Crn1p-null-mutant does not show any phenotype in actin-dependent processes (Heil-Chapdelaine et al., 1998), suggesting that in this organism coronin does not perform an essential function in regulating the actin cytoskeleton. Although lower eukaryotes have one coronin gene, database searches have revealed the existence of several coronins in humans and mice (denoted coronins 1-7) (Okumura et al., 1998;de Hostos, 1999;Rybakin et al., 2004).In macrophages and lymphocytes, coronin 1 concentrates at sites of rearrangement of the cytoskeleton. In lymphocytes, coronin 1 assembles at the immunological synapse formed during activation of T-cells (Nal et al., 2004). In mouse macrophages, coronin 1 accumulates during phagocytosis at the cytosolic face of phagosomes and is actively retained by pathogenic mycoba...