Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.

Neurofibromin is a large and multifunctional protein encoded by the tumor suppressor gene NF1, mutations of which cause the tumor predisposition syndrome neurofibromatosis type 1 (NF1). Over the last three decades, studies of neurofibromin structure, interacting partners, and functions have shown that it is involved in several cell signaling pathways, including the Ras/MAPK, Akt/mTOR, ROCK/LIMK/cofilin, and cAMP/PKA pathways, and regulates many fundamental cellular processes, such as proliferation and migration, cytoskeletal dynamics, neurite outgrowth, dendritic-spine density, and dopamine levels. The crystallographic structure has been resolved for two of its functional domains, GRD (GAP-related (GTPase-activating protein) domain) and SecPH, and its post-translational modifications studied, showing it to be localized to several cell compartments. These findings have been of particular interest in the identification of many therapeutic targets and in the proposal of various therapeutic strategies to treat the symptoms of NF1. In this review, we provide an overview of the literature on neurofibromin structure, function, interactions, and regulation and highlight the relationships between them.

TolA, -B, -Q, and -R proteins are involved in maintaining the cell envelope integrity of Escherichia coli; they have been parasitized by the group A colicins and the single strand DNA of some filamentous bacteriophages to permit them to enter the cells. TolA and TolR are anchored to the inner membrane by a single transmembrane domain, TolQ is an integral membrane protein with three transmembrane segments, and TolB has recently been found to be periplasmic although it is partially membrane-associated. The latter result suggests that TolB might interact with membrane proteins. Other lines of evidence favor the existence of a Tol complex. To further characterize this complex, we investigated which proteins interact with TolB. For this purpose, two different methods were used. First, we took advantage of the existence of a tagged TolB (TolBep) to perform immunoprecipitation under native conditions in order to preserve the putative associations of TolBep with other proteins. Secondly, in vivo cross-linking experiments with formaldehyde were performed. These two approaches led to the same result and demonstrated for the first time that a component of the Tol system, TolB, interacts with a protein located in the outer membrane, the peptidoglycan-associated lipoprotein.

The Saccharomyces cerevisiae alpha-pheromone receptor, a polytopic, G protein-coupled, membrane protein, is internalized after binding of alpha-factor. Mutational analysis suggested that the first 39 residues of the receptor's cytoplasmic tail carries sufficient information for internalization. A point mutation in one of these 39 residues, K337 to R337, renders the receptor nonfunctional for endocytosis. Other residues, D335 and S338, contribute to the efficiency of internalization. When the sequence DAKSS is added onto a severely truncated receptor, endocytosis of the receptor is restored, showing that this sequence functions to mediate or to signal interaction with the endocytic machinery. Analysis of pheromone response and recovery in strains expressing mutant receptors suggests that receptor internalization is not important for response but contributes to recovery from pheromone.

Significance: This study provides evidence of a physical interaction between neurofibromin, an Ras-GTPase activating protein, and a G protein-coupled receptor (GPCR), the serotonin 5 hydroxytryptamine 6 (5-HT₆) receptor. It also shows that constitutive activity of a GPCR depends on its association with GPCR-interacting proteins. Finally, it suggests that the 5-HT₆receptor may be considered as a potentially relevant therapeutic target to correct some neurofibromatosis type 1-related cognitive deficits, a genetic disorder caused by mutations in the gene encoding neurofibromin.