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Query: UNIPROT:P06889 (Mol)
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Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit of the channel that guides proteins across the membrane of the endoplasmic reticulum. The split-ubiquitin technique was used to determine whether Ssh1p serves as an additional receptor for signal sequences in vivo. We measured the interactions between the N(ub)-labeled Ssh1p and C(ub)-translocation substrates bearing four different signal sequences. The so-determined interaction profile of Ssh1p was compared with the signal sequence interaction profile of the correspondingly modified N(ub)-Sec61p. The assay reveals interactions of Ssh1p with the signal sequences of Kar2p and invertase, whereas Sec61p additionally interacts with the signal sequences of Mfalpha1 and carboxypeptidase Y. The measured physical proximity between Ssh1p and the beta-subunit of the signal sequence recognition particle receptor confirms our hypothesis that Ssh1p is directly involved in the cotranslational translocation of proteins across the membrane of the endoplasmic reticulum.
Mol Biol Cell 2002 Jul
PMID:Recognition of a subset of signal sequences by Ssh1p, a Sec61p-related protein in the membrane of endoplasmic reticulum of yeast Saccharomyces cerevisiae. 1213 63

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.
Mol Biol Cell 2002 Jul
PMID:Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. 1213 85

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association.
Mol Biol Cell 2002 Aug
PMID:Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p. 1218 49

Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.
Mol Biol Cell 2002 Sep
PMID:ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization. 1222 Nov 17

The Saccharomyces cerevisiae genome contains five genes encoding P-type ATPases that are potential aminophospholipid translocases (APTs): DRS2, NEO1, and three uncharacterized open reading frames that we have named DNF1, DNF2, and DNF3 for DRS2/NEO1 family. NEO1 is the only essential gene in APT family and seems to be functionally distinct from the DRS2/DNF genes. The drs2Delta dnf1Delta dnf2Delta dnf3Delta quadruple mutant is inviable, although any one member of this group can maintain viability, indicating that there is a substantial functional overlap between the encoded proteins. We have previously implicated Drs2p in clathrin function at the trans-Golgi network. In this study, we constructed strains carrying all possible viable combinations of null alleles from this group and analyzed them for defects in protein transport. The drs2Delta dnf1Delta mutant grows slowly, massively accumulates intracellular membranes, and exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole. Transport of carboxypeptidase Y to the vacuole is also perturbed, but to a lesser extent. In addition, the dnf1Delta dnf2Delta dnf3Delta mutant exhibits a defect in recycling of GFP-Snc1p in the early endocytic-late secretory pathways. Drs2p and Dnf3p colocalize with the trans-Golgi network marker Kex2p, whereas Dnf1p and Dnf2p seem to localize to the plasma membrane and late exocytic or early endocytic membranes. We propose that eukaryotes express multiple APT subfamily members to facilitate protein transport in multiple pathways.
Mol Biol Cell 2002 Sep
PMID:An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system. 1222 Nov 23

Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild-type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH-specific reagent 1-chloro-2,4-dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.
Mol Microbiol 2002 Nov
PMID:Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae. 1241 Aug 42

Some beneficial effects of ACE inhibitors are attributed to potentiation of bradykinin's actions exerted through its B2 receptor. We investigated them on cultured cells transfected or constitutively expressing both ACE and B2 receptor. The potentiation of bradykinin was indirect and attributed to a crosstalk induced between enzyme and receptor via ACE, a heterodimer formation. While looking for endogenous activators, we investigated the split products of angiotensin I (Ang) Ang 1-9 and 1-7, peptides released by enzymes of human atria and ventricle. Ang 1-9 was liberated by a cathepsin A-type enzyme, Ang 1-7 by a different metallopeptidase-protease. Cathepsin A's presence in heart tissue was shown by deamidating enkephalinamide substrate, by immunoprecipitation and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. Ang 1-9 and Ang 1-7 potentiated the effect of an ACE-resistant bradykinin analogue on the B2 receptor in transfected cells expressing human ACE and B2, and in human endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and NO release by bradykinin. NO liberation by bradykinin from endothelial cells was potentiated at 10nmol/L concentration by Ang 1-9 and Ang 1-7; at higher concentrations, Ang 1-9 was significantly more active. Both peptides had little activity in absence of bradykinin or ACE. Ang 1-9 and 1-7 potentiated bradykinin action on its B2 receptor at much lower concentrations than their IC50 values with ACE. They probably induce conformational changes in the ACE/B2 receptor complex via interaction with ACE.
J Mol Cell Cardiol 2002 Dec
PMID:Products of angiotensin I hydrolysis by human cardiac enzymes potentiate bradykinin. 1250 55

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between the phenotype and cre expression, aberrant phenotypes always co-segregate with the transgene, which strongly suggests a correlation. The severity of the phenotype does not correlate with the level of steady-state mRNA in mature leaves, but with the timing of cre expression during organogenesis. The early onset of cre expression in tomato is correlated with a more severe phenotype and with higher germinal transmission frequencies of site-specific deletions. No aberrant phenotype was observed when a tissue-specific phaseolin promoter was used to drive the cre gene. The data suggest that for the application of recombinases in plants, expression is best limited to specific tissues and a short time frame.
Plant Mol Biol 2003 Jan
PMID:Cre recombinase expression can result in phenotypic aberrations in plants. 1260 84

A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described. One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis. Thirty three genomic sequences governing expression of a reporter protein have been isolated. Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns. Another isolated DNA fragment encoded a homolog of the S. cerevisiae V ps10p. Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium.
Mol Biol (Mosk)
PMID:[A novel approach to isolation and functional characterization of genomic DNA from the methylotrophic yeast Hansenula polymorpha]. 1262 50

Galactosialidosis is an autosomal recessive lysosomal storage disease caused by a combined deficiency of lysosomal beta-galactosidase and neuraminidase as a result of a primary defect in the protective protein/cathepsin A (PPCA). We report the first 2 Dutch cases of early infantile galactosialidosis, both presenting with neonatal ascites. The defect was identified in urine, leukocytes, and fibroblasts. Residual activity was determined with a modified assay for cathepsin A and was <5% in leukocytes and <1% in fibroblasts. Histological examination of the placenta in case 1 showed extensive vacuolization in all cell types. Northern blot analysis of RNA isolated from the patients' cultured fibroblasts showed substantially decreased levels of the PPCA transcript, which nevertheless had the correct size of 2 kb. Mutation analysis of both mRNA and genomic DNA from the patients identified two novel mutations in the PPCA locus. Case 1 was a compound heterozygote, with a single missense mutation in one allele, which resulted in Gly57Ser amino acid substitution, and a single C insertion at nucleotide position 899 in the second allele, which gave rise to a frame shift and premature termination codon. Case 2 was homozygous for the same C899 insertion found in case 1.
Mol Genet Metab 2003 Mar
PMID:New mutations in two Dutch patients with early infantile galactosialidosis. 1264 68


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