The fluid mosaic membrane model proved to be a very useful hypothesis in explaining many, but certainly not all, phenomena taking place in biological membranes. New experimental data show that the compartmentalization of membrane components can be as important for effective signal transduction as is the fluidity of the membrane. In this work, we pay tribute to the Singer-Nicolson model, which is near its 30th anniversary, honoring its basic features, ''mosaicism'' and ''diffusion,'' which predict the interspersion of proteins and lipids and their ability to undergo dynamic rearrangement via Brownian motion. At the same time, modifications based on quantitative data are proposed, highlighting the often genetically predestined, yet flexible, multilevel structure implementing a vast complexity of cellular functions. This new ''dynamically structured mosaic model'' bears the following characteristics: emphasis is shifted from fluidity to mosaicism, which, in our interpretation, means nonrandom codistribution patterns of specific kinds of membrane proteins forming smallscale clusters at the molecular level and large-scale clusters (groups of clusters, islands) at the submicrometer level. The cohesive forces, which maintain these assemblies as principal elements of the membranes, originate from within a microdomain structure, where lipid-lipid, protein-protein, and protein-lipid interactions, as well as sub-and supramembrane (cytoskeletal, extracellular matrix, other cell) effectors, many of them genetically predestined, play equally important roles. The concept of fluidity in the original model now is interpreted as permissiveness of the architecture to continuous, dynamic restructuring of the molecular-and higherlevel clusters according to the needs of the cell and as evoked by the environment.

Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.Protein ubiquitylation has emerged as a versatile regulatory strategy (reviewed in Ref. 1). In its best characterized role as a signal for proteasomal degradation, productive recognition of ubiquitylated substrates is shown to minimally require a tetraubiquitin chain (2). Alternatively, studies in yeast attribute to monoubiquitylation an intrinsic capacity to target substrates both for internalization at the plasma membrane and sorting at multivesicular bodies toward destruction in the vacuole (reviewed in Ref. 3). With subsequent identification of ubiquitin binding activities, such as the UIM, 1 a rationale for ubiquitindependent recognition of substrates has materialized (4 -7). In higher eukaryotic systems, ubiquitylation of cell-surface receptors, likewise, correlates with their down-regulation via orthologous trafficking pathways that employ counterparts conserved from yeast (8 -11). Ligand-activated ubiquitylation of EGFR, as well as other RTKs, is mediated by c-Cbl (12-14).Whether or not EGFR ubiquitylation is sufficient for its internalization remains an open question. Likewise, although it is clear that each endocytosed receptor is conjugated to several molecules of ubiquitin, it is currently unknown to which extent branching of the EGFR-conjugated ubiquitins occurs in living cells. Here we present evidence indicating that the action of c-Cbl is limited to the addition of monomeric ubiquitins, and these are sufficient for receptor endocytosis and degradation. EXPERIMENTAL PROCEDURESReagents and Antibodies-Unless indicated, reagents were purchased from Sigma. E1 was from Affiniti (Mamhead, Exeter, UK), and rabbit reticulocyte lysate from Promega (Madison, WI). The 528-IgG antibody was isolated from hybridomas and a Fab fragment prepared and labeled with Cy3. An antibody to EGFR was from Alexis (San Diego, CA). Anti-EEA1 mouse antibody was from Transduction Laboratories (Lexington, KY). Fluorescently labeled antibodies were purchased from Jackson ImmunoResearch (West Grove, PA).Co...