Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.     

A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast.

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.     

The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP-1 complex: evidence for functional interaction with clathrin at the Golgi complex.

Clathrin-associated protein (AP) complexes have been implicated in the assembly of clathrin coats and the selectivity of clathrin-mediated protein transport processes. We have identified a yeast gene, APS1, encoding a homolog of the small (referred to herein as sigma) subunits of the mammalian AP-1 complex. Sequence comparisons have shown that Aps1p is more similar to the sigma subunit of the Golgi-localized mammalian AP-1 complex than Aps2p, which is more related to the plasma membrane AP-2 sigma subunit. Like their mammalian counterparts, Aps1p and Aps2p are components of distinct, large (> 200 kDa) complexes and a significant portion of the Aps proteins co-fractionate with clathrin-coated vesicles during gel filtration chromatography. Unexpectedly, even though the evolutionary conservation of AP small subunits is substantial (50% identity between mammalian and yeast proteins), disruptions of APS1 (aps1 delta) and APS2 (aps2 delta), individually or in combination, elicit no detectable mutant phenotypes. These data indicate that the Aps proteins are not absolutely required for clathrin-mediated selective protein transport in cells expressing wild type clathrin. However, aps1 delta accentuated the slow growth and alpha-factor pheromone maturation defect of cells carrying a temperature-sensitive allele of clathrin heavy chain (Chc) (chc1-ts). In contrast, aps1 delta did not influence the effects of chc1-ts on vacuolar protein sorting or receptor-mediated endocytosis. The aps2 delta mutation resulted in a slight effect on chc1-ts cell growth but had no additional effects. The growth defect of cells completely lacking Chc was compounded by aps1 delta but not aps2 delta. These results comprise evidence that Aps1p is involved in a subset of clathrin functions at the Golgi apparatus. The effect of aps1 delta on cells devoid of clathrin function suggests that Aps1p also participates in clathrin-independent processes.     

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.     

Suppressors of YCK-encoded yeast casein kinase 1 deficiency define the four subunits of a novel clathrin AP-like complex.

In Saccharomyces cerevisiae, the redundant YCK1 and YCK2 genes (Yeast Casein Kinase 1) are required for viability. We describe here the molecular analysis of four mutations that eliminate the requirement for Yck activity. These mutations alter proteins that resemble the four subunits of clathrin adaptors (APs), with highest sequence similarity to those of the recently identified AP-3 complex. The four yeast subunits are associated in a high-molecular-weight complex. These proteins have no essential function and are not redundant for function with other yeast AP-related proteins. Combination of suppressor mutations with a clathrin heavy chain mutation (chc1-ts) confers no synthetic growth defects. However, a yck(ts) mutation shows a strong synthetic growth defect with chc1-ts. Moreover, endocytosis of Ste3p is dramatically decreased in yck(ts) cells and is partially restored by the AP suppressor mutations. These results suggest that vesicle trafficking at the plasma membrane requires the activity of Yck protein kinases, and that the new AP-related complex may participate in this process.     

Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast

Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.     

Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.     

Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake

Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization.     

Ubiquitination of the yeast a-factor receptor

The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination. Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor. We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity. Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct. Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F. Sprague, Jr., 1993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J. Rohrer, F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor. In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions. Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis. Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system. In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cell. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis. To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.     

Recycling of the yeast a-factor receptor

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.     

Clathrin and two components of the COPII complex, Sec23p and Sec24p, could be involved in endocytosis of the Saccharomyces cerevisiae maltose transporter

The Saccharomyces cerevisiae maltose transporter is a 12-transmembrane segment protein that under certain physiological conditions is degraded in the vacuole after internalization by endocytosis. Previous studies showed that endocytosis of this protein is dependent on the actin network, is independent of microtubules, and requires the binding of ubiquitin. In this work, we attempted to determine which coat proteins are involved in this endocytosis. Using mutants defective in the heavy chain of clathrin and in several subunits of the COPI and the COPII complexes, we found that clathrin, as well as two cytosolic subunits of COPII, Sec23p and Sec24p, could be involved in internalization of the yeast maltose transporter. The results also indicate that endocytosis of the maltose transporter and of the alpha-factor receptor could have different requirements.     

Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis

Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants.     

Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin

Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference-based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.     

Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.     

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.     

The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.     

Notch down-regulation by endocytosis is essential for pigment cell determination and survival in the Drosophila retina

The clathrin heavy chain is a fundamental element in endocytosis and therefore, in the internalization of several cell-surface receptors through which cells interact with their environment. Here we show that the only non-lethal mutant allele of the clathrin heavy chain identified to date in metazoans, the Drosophila Chc⁴, involves the substitution of a residue at the knee region of the molecule that impairs clathrin-dependent endocytosis. We have investigated the consequences of this endocytic defect in Drosophila retinal development and found that it produces an inhibition of programmed cell death in the retinal lattice, followed by widespread death of interommatidial pigment cells once retinal development has been completed. Through genetic interactions and transgenic analyses, we show that Chc⁴ phenotypes are caused by a Notch receptor gain-of-function, providing a dramatic example of the importance of Notch down-regulation by endocytosis. An increase in Notch signaling is also observed in Drosophila wings in response to the mutant clathrin, suggesting that Notch levels are controlled by clathrin-dependent endocytosis. We discuss the implications of these findings for current models on eye-development and for the role of endocytosis in Notch signaling.     

Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor

Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.     

Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae.

STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae. To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization. We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed.     

A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the alpha-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.