Distinct functional surface regions on ubiquitin.

The characterized functions of the highly conserved polypeptide ubiquitin are to target proteins for proteasome degradation or endocytosis. The formation of a polyubiquitin chain of at least four units is required for efficient proteasome binding. By contrast, monoubiquitin serves as a signal for the endocytosis of plasma membrane proteins. We have defined surface residues that are important for ubiquitin's vital functions in Saccharomyces cerevisiae. Surprisingly, alanine scanning mutagenesis showed that only 16 of ubiquitin's 63 surface residues are essential for vegetative growth in yeast. Most of the essential residues localize to two hydrophobic clusters that participate in proteasome recognition and/or endocytosis. The others reside in or near the tail region, which is important for conjugation and deubiquitination. We also demonstrate that the essential residues comprise two distinct functional surfaces: residues surrounding Phe(4) are required for endocytosis, whereas residues surrounding Ile(44) are required for both endocytosis and proteasome degradation.

Multiple roles for Rsp5p-dependent ubiquitination at the internalization step of endocytosis.

Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway. The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the alpha-factor receptor (Ste2p). Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis. Temperature-sensitive rsp5 mutant cells were defective in the internalization of alpha-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells. Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p. Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitin-dependent and -independent endocytosis signals. Constitutive Ste2p-ubiquitin internalization and fluid-phase endocytosis also required active ubiquitination machinery, including Rsp5p. These observations indicate that Rsp5p-dependent ubiquitination of a trans-acting protein component of the endocytosis machinery is required for the internalization step of endocytosis.

Protein regulation by monoubiquitin.

Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitylated proteins by the proteasome, but other functions of ubiquitin have been discovered that do not involve the proteasome. Some proteins are modified by a single ubiquitin or short ubiquitin chains. Instead of sending proteins to their death through the proteasome, monoubiquitylation regulates processes that range from membrane transport to transcriptional regulation.

Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis.

Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast alpha-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on alpha-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

Monoubiquitin carries a novel internalization signal that is appended to activated receptors.

Ubiquitin modification of signal transducing receptors at the plasma membrane is necessary for rapid receptor internalization and downregulation. We have investigated whether ubiquitylation alters a receptor cytoplasmic tail to reveal a previously masked internalization signal, or whether ubiquitin itself carries an internalization signal. Using an alpha-factor receptor-ubiquitin chimeric protein, we demonstrate that monoubiquitin can mediate internalization of an activated receptor that lacks all cytoplasmic tail sequences. Furthermore, fusion of ubiquitin in-frame to the stable plasma membrane protein Pma1p stimulates endocytosis of this protein. Ubiquitin does not carry a functional tyrosine- or di-leucine-based internalization signal. Instead, the three-dimensional structure of the folded ubiquitin polypeptide carries an internalization signal that consists of two surface patches surrounding the critical residues Phe4 and Ile44. We conclude that ubiquitin functions as a novel regulated internalization signal that can be appended to a plasma membrane protein to trigger downregulation.

Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels.

G-protein-coupled receptors and transporters in Saccharomyces cerevisiae are modified with ubiquitin in response to ligand biding. In most cases, the proteasome does not recognize these ubiquitinated proteins. Instead, ubiquitination serves to trigger internalization and degradation of plasma membrane proteins in the lysosome-like vacuole. A number of mammalian receptors and at least one ion channel undergo ubiquitination at the plasma membrane, and this modification is required for their downregulation. Some of these cell-surface proteins appear to be degraded by both the proteasome and lysosomal proteases. Recent evidence indicates that other proteins required for receptor internalization might also be regulated by ubiquitination, suggesting that ubiquitin plays diverse roles in regulating plasma membrane protein activity.

A function for monoubiquitination in the internalization of a G protein-coupled receptor.

Modification of an S. cerevisiae G protein-coupled receptor with ubiquitin is required for its ligand-stimulated internalization. We now demonstrate that monoubiquitination on a single lysine residue is sufficient to signal receptor internalization, a modification distinct from that required for proteasome recognition. Formation of a polyubiquitin chain is not necessary, as demonstrated by the ability of mutant ubiquitins that lack lysine residues to serve as efficient internalization signals. Fusion of ubiquitin in-frame to a receptor that lacks cytoplasmic tail lysines also promotes rapid receptor internalization, indicating that ubiquitin itself and not a specific type of linkage to the receptor acts as an internalization signal. Thus, we have defined a cellular function for monoubiquitination in alpha-factor receptor endocytosis.

Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization.

G protein-coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The alpha-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding alpha-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 alpha-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.

Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins.

The modification of cytosolic proteins with polyubiquitin chains targets them for recognition and degradation by the multisubunit proteolytic particle, the 26S proteasome. Membrane proteins are also substrates for ubiquitination. Integral membrane proteins of the endoplasmic reticulum are ubiquitinated and destroyed by the proteasome. However, it has been shown recently that the ubiquitination of Saccharomyces cerevisiae plasma membrane proteins signals their degradation by the proteolytic system in the lysosome-like vacuole. Ubiquitination of several different classes of cell surface proteins serves as a signal for their entry into the endocytic pathway; this leads to their transport to the vacuole, where they are permanently inactivated by degradation. In yeast, ubiquitin has been implicated as an internalization signal for most, if not all, endogenous plasma membrane proteins that are known to be endocytosed. Ubiquitin-dependent internalization has been best characterized for two proteins: the mating pheromone alpha-factor receptor and the uracil permease. Some mammalian cell surface receptors are also ubiquitinated at the plasma membrane. Ubiquitination machinery is required for ligand-induced endocytosis of the growth hormone receptor, suggesting that ubiquitin-dependent endocytosis and sorting is also an important regulatory process in mammalian cells. Mammalian receptors may also be down-regulated through the degradation of their cytosolic domains by a proteasome-dependent pathway.

End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.

Actin-, myosin- and ubiquitin-dependent endocytosis.

Endocytosis is a general term that is used to describe the internalization of external and plasma membrane molecules into the cell interior. In fact, several different mechanisms exist for the internalization step of this process. In this review we emphasize the work on the actin-dependent pathways, in particular in the yeast Saccharomyces cerevisiae, because several components of the molecular machinery are identified. In this yeast, the analysis of endocytosis in various mutants reveals a requirement for actin, calmodulin, a type I myosin, as well as a number of other proteins that affect actin dynamics. Some of these proteins have homology to proteins in animal cells that are believed to be involved in endocytosis. In addition, the demonstration that ubiquitination of some cell surface molecules is required for their efficient internalization is described. We compare the actin, myosin and ubiquitin requirements for endocytosis with recent results found studying these processes using Dictyostelium discoideum and animal cells.

Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis.

Binding of alpha factor to Ste2p, a G protein-coupled plasma membrane receptor, activates a signal transduction pathway and stimulates endocytosis of the receptor-ligand complex. Ligand binding also induces ubiquitination of the Ste2p cytoplasmic tail. Protein ubiquitination is required for stimulated endocytosis of Ste2p, as internalization is 5- to 15-fold slower in ubc mutants that lack multiple ubiquitin-conjugating enzymes. In a C-terminal truncated form of Ste2p that is rapidly ubiquitinated and endocytosed in response to ligand binding, a single lysine to arginine substitution in its cytoplasmic tail eliminates both ubiquitination and internalization. Thus, ubiquitination of Ste2p itself is required for ligand-stimulated endocytosis. We propose that ubiquitination mediates degradation of receptor-ligand complexes, not via the proteasome, but by acting as a signal for endocytosis leading to subsequent degradation in the lysosome/vacuole.

Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.

Molecular machinery required for protein transport from the endoplasmic reticulum to the Golgi complex.

The cellular machinery responsible for conveying proteins between the endoplasmic reticulum and the Golgi is being investigated using genetics and biochemistry. A role for vesicles in mediating protein traffic between the ER and the Golgi has been established by characterizing yeast mutants defective in this process, and by using recently developed cell-free assays that measure ER to Golgi transport. These tools have also allowed the identification of several proteins crucial to intracellular protein trafficking. The characterization and possible functions of several GTP-binding proteins, peripheral membrane proteins, and an integral membrane protein during ER to Golgi transport are discussed here.

Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro.

The SEC23 gene product (Sec23p) is required for transport of secretory, plasma membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex in Saccharomyces cerevisiae. Molecular cloning and biochemical characterization demonstrate that Sec23p is an 84 kd unglycosylated protein that resides on the cytoplasmic surface of a large structure, possibly membrane or cytoskeleton. Vigorous homogenization of yeast cells or treatment of yeast lysates with reagents that desorb peripheral membrane proteins releases Sec23p in a soluble form. Protein transport from the endoplasmic reticulum to the Golgi in vitro depends upon active Sec23p. Thermosensitive transport in sec23 mutant lysates is restored to normal when a soluble form of wild-type Sec23p is added, providing a biochemical complementation assay for Sec23p function. Gel filtration of yeast cytosol indicates that functional Sec23p is a large oligomer or part of a multicomponent complex.