scholarly journals Two AAA Family Peroxins, PpPex1p and PpPex6p, Interact with Each Other in an ATP-Dependent Manner and Are Associated with Different Subcellular Membranous Structures Distinct from Peroxisomes

1998 ◽  
Vol 18 (2) ◽  
pp. 936-943 ◽  
Author(s):  
Klaas Nico Faber ◽  
John A. Heyman ◽  
Suresh Subramani
Keyword(s):  
Subcellular Fractionation ◽  
Matrix Proteins ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Peroxisome Biogenesis ◽  
Yeast Two Hybrid System ◽  
Cell Biol ◽  
Sensitive Allele ◽  
Two Hybrid

ABSTRACT Two peroxins of the AAA family, PpPex1p and PpPex6p, are required for peroxisome biogenesis in the yeast Pichia pastoris. Cells from the corresponding deletion strains (PpΔpex1and PpΔpex6) contain only small vesicular remnants of peroxisomes, the bulk of peroxisomal matrix proteins is mislocalized to the cytosol, and these cells cannot grow in peroxisome-requiring media (J. A. Heyman, E. Monosov, and S. Subramani, J. Cell Biol. 127:1259–1273, 1994; A. P. Spong and S. Subramani, J. Cell Biol. 123:535–548, 1993). We demonstrate that PpPex1p and PpPex6p interact in an ATP-dependent manner. Genetically, the interaction was observed in a suppressor screen with a strain harboring a temperature-sensitive allele of PpPEX1 and in the yeast two-hybrid system. Biochemially, these proteins were coimmunoprecipitated with antibodies raised against either of the proteins, but only in the presence of ATP. The protein complex formed under these conditions was 320 to 400 kDa in size, consistent with the formation of a heterodimeric PpPex1p-PpPex6p complex. Subcellular fractionation revealed PpPex1p and PpPex6p to be predominantly associated with membranous subcellular structures distinct from peroxisomes. Based on their behavior in subcellular fractionation experiments including flotation gradients and on the fact that these structures are also present in a PpΔpex3strain in which no morphologically detectable peroxisomal remnants have been observed, we propose that these structures are small vesicles. The identification of vesicle-associated peroxins is novel and implies a role for these vesicles in peroxisome biogenesis. We discuss the possible role of the ATP-dependent interaction between PpPex1p and PpPex6p in regulating peroxisome biogenesis events.

scholarly journals Peroxisome Biogenesis Occurs in an Unsynchronized Manner in Close Association with the Endoplasmic Reticulum in Temperature-sensitiveYarrowia lipolyticaPex3p Mutants

2003 ◽  
Vol 14 (3) ◽  
pp. 939-957 ◽  
Author(s):  
Roger A. Bascom ◽  
Honey Chan ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Matrix Protein ◽  
De Novo ◽  
Close Association ◽  
Integral Membrane Protein ◽  
Matrix Proteins ◽  
Temperature Sensitive ◽  
Tubular Structures ◽  
Peroxisome Biogenesis

Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes. Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis. In glucose medium at 16°C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei. The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells. Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16°C. These structures were not detected in wild-type or Pex3p-deficient cells. Their role in peroxisome biogenesis remains unclear. Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery. Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577 ◽  
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

scholarly journals The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane.

1995 ◽  
Vol 131 (6) ◽  
pp. 1517-1527 ◽  
Author(s):  
K G Kozminski ◽  
P L Beech ◽  
J L Rosenbaum
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Intraflagellar Transport ◽  
Temperature Sensitive ◽  
Polystyrene Beads ◽  
Cell Biol ◽  
Flagellar Membrane ◽  
Surface Motility ◽  
Sensitive Allele ◽  
Bidirectional Movement

The Chlamydomonas FLA10 gene was shown to encode a flagellar kinesin-like protein (Walther, Z., M. Vashishtha, and J.L. Hall. 1994. J. Cell Biol. 126:175-188). By using a temperature-sensitive allele of FLA10, we have determined that the FLA10 protein is necessary for both the bidirectional movement of polystyrene beads on the flagellar membrane and intraflagellar transport (IFT), the bidirectional movement of granule-like particles beneath the flagellar membrane (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. (USA). 90:5519-5523). In addition, we have correlated the presence and position of the IFT particles visualized by light microscopy with that of the electron dense complexes (rafts) observed beneath the flagellar membrane by electron microscopy. A role for FLA10 in submembranous or flagellar surface motility is also strongly supported by the immunolocalization of FLA10 to the region between the axonemal outer doublet microtubules and the flagellar membrane.

scholarly journals Functional interactions between the hBRM/hBRG1 transcriptional activators and the pRB family of proteins.

1996 ◽  
Vol 16 (4) ◽  
pp. 1576-1583 ◽  
Author(s):  
B E Strober ◽  
J L Dunaief ◽  
Guha ◽  
S P Goff
Keyword(s):  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Adenocarcinoma Cell Line ◽  
Cycle Progression ◽  
Yeast Two Hybrid System ◽  
Flat Cell ◽  
Two Hybrid

hBRG1 and hBRM are mammalian homologs of the SNF2/SW12 yeast transcriptional activator. These proteins exist in a large multisubunit complex that likely serves to remodel chromatin and, in so doing, facilitates the function of specific transcription factors. The retinoblastoma protein (pRB) inhibits cell cycle progression by repressing transcription of specific growth-related genes. Using the yeast two-hybrid system, we demonstrate that the members of the hBRG1/hBRM family of proteins interact with the pRB family of proteins, which includes pRB, p107, and p130. Interaction between the hBRG1/hBRM family with the pRB family likely influences cellular proliferation, as both hBRG1 and hBRM, but not mutants of these proteins unable to bind to pRB family members, inhibit the formation of drug-resistant colonies when transfected into the SW13 human adenocarcinoma cell line, which lacks endogenous hBRG1 or hBRM. Further, hBRM and two isoforms of hBRG1 induce the formation of flat, growth-arrested cells in a pRB family-dependent manner when introduced into SW13 cells. This flat-cell inducing activity is severely reduced by cotransfection of the wild-type E1A protein and variably reduced by the cotransfection of mutants of E1A that lack the ability to bind to some or all members of the pRB family.

scholarly journals The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner

1994 ◽  
Vol 300 (2) ◽  
pp. 303-307 ◽  
Author(s):  
M Spaargaren ◽  
G A Martin ◽  
F McCormick ◽  
M J Fernandez-Sarabia ◽  
J R Bischoff
Keyword(s):  
Amino Acids ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Downstream Effector ◽  
Yeast Two Hybrid System ◽  
Vitro Binding ◽  
Ras Family ◽  
In Vitro Binding Assay ◽  
Two Hybrid

R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector.

scholarly journals Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences

2020 ◽  
Vol 8 (8) ◽  
pp. 1229
Author(s):  
Herbert J. Santos ◽  
Yoko Chiba ◽  
Takashi Makiuchi ◽  
Saki Arakawa ◽  
Yoshitaka Murakami ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Intestinal Parasite ◽  
Subcellular Fractionation ◽  
Immunofluorescence Assay ◽  
Matrix Proteins ◽  
Desulfovibrio Vulgaris ◽  
Chimeric Proteins ◽  
Atp Sulfurylase ◽  
Sulfate Activation

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.

scholarly journals The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein

2000 ◽  
Vol 74 (5) ◽  
pp. 2073-2083 ◽  
Author(s):  
Etienne Herzog ◽  
Orlene Guerra-Peraza ◽  
Thomas Hohn
Keyword(s):  
Coat Protein ◽  
Protein Domain ◽  
Rice Tungro Bacilliform Virus ◽  
Viral Gene ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Genes Encoding ◽  
Two Hybrid

ABSTRACT Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.

scholarly journals Mutants of the Yeast Yarrowia lipolyticaDefective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

1998 ◽  
Vol 18 (5) ◽  
pp. 2789-2803 ◽  
Author(s):  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Membrane Proteins ◽  
Matrix Proteins ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mutant Cells

ABSTRACT Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A andsrp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A,srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that inY. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54,PEX1, and PEX6 genes.

scholarly journals Dad1p, Third Component of the Duo1p/Dam1p Complex Involved in Kinetochore Function and Mitotic Spindle Integrity

2001 ◽  
Vol 12 (9) ◽  
pp. 2601-2613 ◽  
Author(s):  
Maria Enquist-Newman ◽  
Iain M. Cheeseman ◽  
David Van Goor ◽  
David G. Drubin ◽  
Pamela B. Meluh ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
The Novel ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sensitive Allele ◽  
Two Hybrid ◽  
Kinetochore Function

We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability.

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