Howard Riezman

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.

Linking cargo to vesicle formation: receptor tail interactions with coat proteins.

How soluble cargo molecules concentrate into budding vesicles is the subject of intensive current research. Clathrin-based vesiculation from the plasma membrane and the trans-Golgi network constitutes the best described system that supports this sorting process. Soluble ligands bind to specific transmembrane receptors which have been shown to interact directly with clathrin adaptor complexes, components of clathrin coats. At the same time, these clathrin adaptors facilitate clathrin coat assembly and probably regulate the recruitment of the rest of the coat components. Recent studies have looked at both the interaction of receptor tails with adaptors and the assembly of the clathrin coat. Progress has also been made in elucidating how soluble cargo molecules may be concentrated for exit from the endoplasmic reticulum.

Sorting GPI-anchored proteins

The study of glycosylphosphatidylinositol-anchored-protein sorting has led to some surprising new findings and concepts. Evidence is accumulating that, during their delivery to the surface, different types of plasma membrane protein might be sorted from each other early in this pathway, in the endoplasmic reticulum. Furthermore, membrane-lipid composition and microdomains might have a role in the process of protein sorting in both the secretory and endocytic pathways.

The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi.

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl‐inositol‐anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha‐factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII‐coated, ER‐derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER‐derived transport vesicles.

Actin and fimbrin are required for the internalization step of endocytosis in yeast.

In Saccharomyces cerevisiae, alpha‐factor is internalized by receptor‐mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta‐tubulin and actin mutant strains, we showed that actin plays a direct role in receptor‐mediated internalization of alpha‐factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta‐tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin‐binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha‐factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.

Transcription and translation initiation frequencies of the Escherichia coli lac operon

Abstract We have calculated the number of RNA polymerase molecules transcribing the induced lac operon of Escherichia coli as well as the distance between ribosomes on the proximal z and on the distal a messages † . These values were derived from: (a) rates of induced enzyme synthesis corrected by known turnover numbers to give numbers of enzyme monomers produced per cell per second; (b) the mass of each message derived either directly by hybridization of long-labeled RNA to specific DNA or from the rates of synthesis and decay obtained by hybridization of pulse-labeled RNA; the latter gives about 13 times better resolution; (c) conversion of message mass into a number of funtioning 3′ ends (producing finished polypeptides); size analyses indicate that the z message probably decays by a net directional degradation in the 5′ to 3′ direction. From this the fraction of completed molecules that are full-length and the total number with functional 3′ ends can be derived. (d) Rates of ribosome movement. Calculated values follow. (1) With a 3.3-second interval between transcription initiations, there are 38 molecules of RNA polymerase on z DNA per cell; with 1.7 copies of lac DNA, this gives 23 on each z cistron and five or six on the y and on the a cistrons. This corresponds to a spacing of about 135 nucleotides between polymerases, which is similar to that on the ribosomal cistrons which have only 1.7 seconds separating initiations. (2) There are 20 molecules of β-galactosidase monomer produced per cell per second from 1.1 × 10−4 pg of z RNA. 38% of the message molecules are nascent and not producing enzyme. Half of the completed molecules and 70% of their mass are intact z messages to give 62 molecules of z message containing functional 3′ ends/cell. Ribosomes load onto these messages at 3.2-second intervals to give a ribosomal spacing of 110 nucleotides. An average of 40 ribosomes translate each z message with half of the message molecules translated by more than 28 ribosomes and half by less. (3) Only 2.4 molecules of galactoside acetyltransferase monomer are produced per cell per second from 1.8 × 10−5 pg of a RNA. Only 20% of these molecules are nascent and 80% of the completed ones are intact; this gives 40 molecules of functioning a messages per cell. Ribosomes load to a message at 16-second intervals to give a spacing of 580 nucleotides. These results show that the frequencies of translation initiations can differ for different messages. The faster decay of a compared to z message is consistent with a model in which a ribosome can protect a vulnerable site near the start of a message from inactivation; messages that load less frequently decay faster. The combined net effect of these causally related processes could account for natural polarity (Zabin & Fowler, 1970), i.e. there is a fourfold lower production of enzyme from the a than from the z gene.

Role of Type I Myosins in Receptor-Mediated Endocytosis in Yeast

Type I myosins are thought to drive actin-dependent membrane motility, but the direct demonstration in vivo of their involvement in specific cellular processes has been difficult. Deletion of the genes MYO3 and MYO5, which encode the yeast type I myosins, almost abolished growth. A double-deleted mutant complemented with a MYO5 temperature-sensitive allele (myo5-1) showed a strong defect in the internalization step of receptor-mediated endocytosis, whereas the secretory pathway remained apparently unaffected. Thus, myosin I activity is required for a budding event in endocytosis but not for several other aspects of membrane traffic.

Protein sorting upon exit from the endoplasmic reticulum.

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.

Endocytosis in yeast: Several of the yeast secretory mutants are defective in endocytosis

Yeast cells have been shown to internalize lucifer yellow CH by endocytosis. Internalization of the fluorescent dye is time-, temperature-, and energy-dependent, it is not saturable, and the dye is accumulated in the vacuole. Some of the yeast secretory mutants that accumulate endoplasmic reticulum or Golgi bodies are defective for endocytosis at restrictive temperature, while others are not. All of the mutants that accumulate secretory vesicles are defective for endocytosis. These results suggest that efficient transport of proteins from the endoplasmic reticulum to the Golgi apparatus and from the Golgi to secretory vesicles is not necessary for endocytosis. In contrast, endocytosis may be obligatorily coupled with the latest steps of secretion.

Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells.

The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B1, an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1,upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3H]sphinganine orl-3-[3H]serine demonstrated thatuog1 conferred fumonisin B1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids ofuog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB1, and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays ofuog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that theN-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.