In Vivo Role for Actin-regulating Kinases in Endocytosis and Yeast Epsin Phosphorylation

The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis. Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence [L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines. Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in a pan1-20 ENT1(EE) double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p-Ent1p complex. Interestingly, pan1-20 ent2 Delta but not pan1-20 ent1 Delta double mutants had improved growth and endocytosis over the pan1-20 mutant. We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects. Consistent with synthetic growth defects of pan1-20 ENT1(EE) cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes. These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis. These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions.     

EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.     

Negative regulation of yeast Eps15-like Arp2/3 complex activator, Pan1p, by the Hip1R-related protein, Sla2p, during endocytosis

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites approximately 25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification-tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2DeltaCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.     

Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis

The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Delta, and end3Delta mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.     

The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.     

Cooperative regulation of endocytic vesicle transport by yeast Eps15-like protein Pan1p and epsins

Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.     

Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1DeltaPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5-Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.     

Scd5p mediates phosphoregulation of actin and endocytosis by the type 1 phosphatase Glc7p in yeast

Pan1p plays essential roles in both actin and endocytosis in yeast. It interacts with, and regulates the function of, multiple endocytic proteins and actin assembly machinery. Phosphorylation of Pan1p by the kinase Prk1p down-regulates its activity, resulting in disassembly of the endocytic vesicle coat complex and termination of vesicle-associated actin polymerization. In this study, we focus on the mechanism that acts to release Pan1p from phosphorylation inhibition. We show that Pan1p is dephosphorylated by the phosphatase Glc7p, and the dephosphorylation is dependent on the Glc7p-targeting protein Scd5p, which itself is a phosphorylation target of Prk1p. Scd5p links Glc7p to Pan1p in two ways: directly by interacting with Pan1p and indirectly by interacting with the Pan1p-binding protein End3p. Depletion of Glc7p from the cells causes defects in cell growth, actin organization, and endocytosis, all of which can be partially suppressed by deletion of the PRK1 gene. These results suggest that Glc7p antagonizes the activity of the Prk1p kinase in regulating the functions of Pan1p and possibly other actin- and endocytosis-related proteins.     

Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.     

Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.     

Phosphoregulation of Arp2/3-dependent actin assembly during receptor-mediated endocytosis

In both yeast and mammals, endocytic internalization is accompanied by a transient burst of actin polymerization. The yeast protein kinases Prk1p and Ark1p, which are related to the mammalian proteins GAK and AAK1, are key regulators of this process. However, the molecular mechanism(s) by which they regulate actin assembly at endocytic sites have not yet been determined. The Eps15-like yeast protein Pan1p is a Prk1p substrate that is essential for endocytic internalization and for proper actin organization. Pan1p is an Arp2/3 activator and here we show that this activity is dependent on F-actin binding. Mutation of all 15 Prk1p-targeted threonines in Pan1p to alanines mimicked the ark1Delta prk1Delta phenotype, demonstrating that Pan1p is a key Prk1p target in vivo. Moreover, phosphorylation by Prk1p inhibited the ability of Pan1p to bind to F-actin and to activate the Arp2/3 complex, thereby identifying the endocytic phosphoregulation mechanism of Prk1p. We conclude that Prk1p phosphorylation of Pan1p shuts off Arp2/3-mediated actin polymerization on endocytic vesicles, allowing them to fuse with endosomes.     

Negative regulation of the actin-regulating kinase Prk1p by patch localization-induced autophosphorylation

The Prk1 family of protein kinases are important regulators of endocytosis and actin cytoskeleton in some eukaryotic cells. In budding yeast, Prk1p phosphorylates numerous endocytic proteins including Pan1p and Sla1p. Prk1p has been observed to undergo autophosphorylation in vivo . In this study, we determined the sites and underlying role of the autophosphorylation. Two sites located in the noncatalytic region were identified to be the autophosphorylation sites. When the sites were mutated, the non-autophosphorylatable Prk1p phosphorylated Pan1p and Sla1p more efficiently than the wild-type kinase, suggesting a negative effect of the autophosphorylation. In addition, the dynamic properties of actin and the coat complex were also altered in the autophosphorylation mutant cells. Interestingly, the autophosphorylation of Prk1p was dependent on cortical localization of the kinase and could be induced by phosphorylated Sla1p. These results suggest that the autophosphorylation of Prk1p may represent a feedback mechanism possibly involved in fine-tuning the pace of progression during actin-coupled endocytosis.     

An InCytes from MBC Selection: Polarized Growth in Budding Yeast in the Absence of a Localized Formin

Polarity is achieved partly through the localized assembly of the cytoskeleton. During growth in budding yeast, the bud cortex and neck localized formins Bni1p and Bnr1p nucleate and assemble actin cables that extend along the bud-mother axis, providing tracks for secretory vesicle delivery. Localized formins are believed to determine the location and polarity of cables, hence growth. However, yeast expressing the nonlocalized actin nucleating/assembly formin homology (FH) 1-FH2 domains of Bnr1p or Bni1p as the sole formin grow well. Although cables are significantly disorganized, analysis of directed transport of secretory vesicles is still biased toward the bud, reflecting a bias in correctly oriented cables, thereby permitting polarized growth. Myosin II, localized at the bud neck, contributes to polarized growth as a mutant unable to interact with F-actin further compromises growth in cells with an unlocalized formin but not with a localized formin. Our results show that multiple mechanisms contribute to cable orientation and polarized growth, with localized formins and myosin II being two major contributors.     

Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.     

The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton

Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2- H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament- associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis.     

Scd5p and clathrin function are important for cortical actin organization,endocytosis, and localization of sla2p in yeast

SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but an scd5-Delta338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Delta338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Delta338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars. scd5-Delta338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Delta338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells.     

A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.     

Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.