scholarly journals Pex11-related Proteins in Peroxisome Dynamics: A Role for the Novel Peroxin Pex27p in Controlling Peroxisome Size and Number in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (10) ◽  
pp. 4089-4102 ◽  
Author(s):  
Yuen Yi C. Tam ◽  
Juan C. Torres-Guzman ◽  
Franco J. Vizeacoumar ◽  
Jennifer J. Smith ◽  
Marcello Marelli ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Transcriptome Profiling ◽  
The Novel ◽  
Yeast Two Hybrid ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Related Proteins ◽  
Two Hybrid

Transcriptome profiling identified the gene PEX25 encoding Pex25p, a peroxisomal membrane peroxin required for the regulation of peroxisome size and maintenance in Saccharomyces cerevisiae. Pex25p is related to a protein of unknown function encoded by the open reading frame, YOR193w, of the S. cerevisiae genome. Yor193p is a peripheral peroxisomal membrane protein that exhibits high sequence similarity not only to Pex25p but also to the peroxisomal membrane peroxin Pex11p. Unlike Pex25p and Pex11p, Yor193p is constitutively expressed in wild-type cells grown in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for the YOR193w gene show a few enlarged peroxisomes. Peroxisomes are greatly enlarged in cells harboring double deletions of the YOR193w and PEX25 genes, the YOR193w and PEX11 genes, and the PEX25 and PEX11 genes. Yeast two-hybrid analyses showed that Yor193p interacts with Pex25p and itself, Pex25p interacts with Yor193p and itself, and Pex11p interacts only with itself. Overexpression of YOR193w, PEX25, or PEX11 led to peroxisome proliferation and the formation of small peroxisomes. Our data suggest a role for Yor193p, renamed Pex27p, in controlling peroxisome size and number in S. cerevisiae.

scholarly journals Pex30p, Pex31p, and Pex32p Form a Family of Peroxisomal Integral Membrane Proteins Regulating Peroxisome Size and Number in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (2) ◽  
pp. 665-677 ◽  
Author(s):  
Franco J. Vizeacoumar ◽  
Juan C. Torres-Guzman ◽  
David Bouard ◽  
John D. Aitchison ◽  
Richard A. Rachubinski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Sequence Similarity ◽  
Integral Membrane Proteins ◽  
Peroxisome Proliferation ◽  
Hypothetical Proteins ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Yeast Yarrowia Lipolytica

The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p—now designated Pex30p, Pex31p, and Pex32p, respectively—together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.

scholarly journals YHR150w and YDR479c encode peroxisomal integral membrane proteins involved in the regulation of peroxisome number, size, and distribution in Saccharomyces cerevisiae

2003 ◽  
Vol 161 (2) ◽  
pp. 321-332 ◽  
Author(s):  
Franco J. Vizeacoumar ◽  
Juan C. Torres-Guzman ◽  
Yuen Yi C. Tam ◽  
John D. Aitchison ◽  
Richard A. Rachubinski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Buoyant Density ◽  
Integral Membrane Proteins ◽  
Hypothetical Proteins ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Wild Type Phenotype ◽  
Yeast Yarrowia Lipolytica

The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid–containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (12) ◽  
pp. 5801-5812
Author(s):  
R A Preston ◽  
M F Manolson ◽  
K Becherer ◽  
E Weidenhammer ◽  
D Kirkpatrick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Zinc Finger ◽  
Genomic Library ◽  
Sequence Similarity ◽  
Carboxypeptidase Y ◽  
Lag Phase ◽  
Significant Similarity ◽  
Reading Frame ◽  
Isolation And Characterization

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

scholarly journals Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

1996 ◽  
Vol 16 (2) ◽  
pp. 593-602 ◽  
Author(s):  
R Candau ◽  
P A Moore ◽  
L Wang ◽  
N Barlev ◽  
C Y Ying ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Sequence Similarity ◽  
Adaptor Proteins ◽  
Yeast Two Hybrid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Evolutionarily Conserved ◽  
Human Proteins ◽  
Two Hybrid

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

scholarly journals MDT-28/PLIN-1 mediates lipid droplet-microtubule interaction via DLC-1 in Caenorhabditis elegans

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kang Xie ◽  
Peng Zhang ◽  
Huimin Na ◽  
Yangli Liu ◽  
Hong Zhang ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Metabolic Diseases ◽  
Genetic Screen ◽  
Dynein Light Chain ◽  
Related Gene ◽  
Yeast Two Hybrid ◽  
Forward Genetic Screen ◽  
Related Proteins ◽  
Two Hybrid ◽  
Hybrid Screening

Abstract Ectopic lipid accumulation in lipid droplets (LD) has been linked to many metabolic diseases. In this study, DHS-3::GFP was used as a LD marker in C. elegans and a forward genetic screen was carried out to find novel LD regulators. There were 140 mutant alleles identified which were divided into four phenotypic categories: enlarged, aggregated, aggregated and small, and decreased. After genetic mapping, mutations in three known LD regulatory genes (maoc-1, dhs-28, daf-22) and a peroxisome-related gene (acox-3) were found to enlarge LDs, demonstrating the reliability of using DHS-3 as a living marker. In the screen, the cytoskeleton protein C27H5.2 was found to be involved in LD aggregation, as was the LD resident/structure-like protein, MDT-28/PLIN-1. Using yeast two-hybrid screening and pull-down assays, MDT-28/PLIN-1 was found to bind to DLC-1 (dynein light chain). Fluorescence imaging confirmed that MDT-28/PLIN-1 mediated the interaction between DHS-3 labeled LDs and DLC-1 labeled microtubules. Furthermore, MDT-28/PLIN-1 was directly bound to DLC-1 through its amino acids 1–210 and 275–415. Taken together, our results suggest that MDT-28/PLIN-1 is involved in the regulation of LD distribution through its interaction with microtubule-related proteins.

scholarly journals The Peroxin Pex19p Interacts with Multiple, Integral Membrane Proteins at the Peroxisomal Membrane

2000 ◽  
Vol 149 (6) ◽  
pp. 1171-1178 ◽  
Author(s):  
William B. Snyder ◽  
Antonius Koller ◽  
A. Jobu Choy ◽  
Suresh Subramani
Keyword(s):  
Membrane Proteins ◽  
Multiple Integral ◽  
Integral Membrane Proteins ◽  
Peroxisome Biogenesis ◽  
Yeast Two Hybrid ◽  
Peroxisomal Membrane ◽  
Site Of Action ◽  
Peroxisome Membrane ◽  
Two Hybrid ◽  
New Protein

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.

Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1444-1444
Author(s):  
Denisa Urban ◽  
Ling Li ◽  
James Wasmuth ◽  
Hilary Christensen ◽  
John Parkinson ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Human Platelets ◽  
Multiprotein Complex ◽  
Yeast Two Hybrid ◽  
Granule Formation ◽  
Reading Frame ◽  
Binding Partner ◽  
Library Screen ◽  
Two Hybrid ◽  
Granule Release

Abstract Abstract 1444 Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis. Disclosures: No relevant conflicts of interest to declare.

Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)

2002 ◽  
Vol 361 (3) ◽  
pp. 443-450 ◽  
Author(s):  
Alesia M. HRUSKA-HAGEMAN ◽  
John A. WEMMIE ◽  
Margaret P. PRICE ◽  
Michael J. WELSH
Keyword(s):  
Ion Channel ◽  
Mammalian Cells ◽  
Hippocampal Neurons ◽  
Sequence Similarity ◽  
Expression System ◽  
Pdz Domain ◽  
Mammalian Brain ◽  
Na Channel ◽  
Yeast Two Hybrid ◽  
Two Hybrid

Neuronal members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of cation channels include the mammalian brain Na+ channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related αENaC or βENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.

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