scholarly journals Recycling of proteins from the Golgi compartment to the ER in yeast.

1990 ◽  
Vol 111 (2) ◽  
pp. 369-377 ◽  
Author(s):  
N Dean ◽  
H R Pelham
Keyword(s):  
Fusion Protein ◽  
Recognition System ◽  
Dependent Manner ◽  
Terminal Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Subcellular Organelles ◽  
Er Retention ◽  
Golgi Compartment ◽  
Carboxyl Terminal

In the yeast Saccharomyces cerevisiae, the carboxyl terminal sequence His-Asp-Glu-Leu (HDEL) has been shown to function as an ER retention sequence (Pelham, H. R. B., K. G. Hardwick, and M. J. Lewis. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1757-1762). To examine the mechanism of retention of soluble ER proteins in yeast, we have analyzed the expression of a preproalpha factor fusion protein, tagged at the carboxyl terminus with the HDEL sequence. We demonstrate that this fusion protein, expressed in vivo, accumulates intracellularly as a precursor containing both ER and Golgi-specific oligosaccharide modifications. The Golgi-specific carbohydrate modification, which occurs in a SEC18-dependent manner, consists of alpha 1-6 mannose linkages, with no detectable alpha 1-3 mannose additions, indicating that the transit of the HDEL-tagged fusion protein is confined to an early Golgi compartment. Results obtained from the fractionation of subcellular organelles from yeast expressing HDEL-tagged fusion proteins suggest that the Golgi-modified species are present in the ER. Overexpression of HDEL-tagged preproalpha factor results in the secretion of an endogenous HDEL-containing protein, demonstrating that the HDEL recognition system can be saturated. These results support the model in which the retention of these proteins in the ER is dependent on their receptor-mediated recycling from the Golgi complex back to the ER.

scholarly journals Fibulin-5 interacts with fibrillin-1 molecules and microfibrils

2005 ◽  
Vol 388 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Lyle J. FREEMAN ◽  
Amanda LOMAS ◽  
Nigel HODSON ◽  
Michael J. SHERRATT ◽  
Kieran T. MELLODY ◽  
...  
Keyword(s):  
Molecular Interactions ◽  
Elastic Fibre ◽  
Dependent Manner ◽  
Terminal Sequence ◽  
Fibre Formation ◽  
Fibrillin 1 ◽  
Dose Dependent

Fibulin-5 plays an important role in elastic fibre formation in vivo. We have investigated the molecular interactions between fibulin-5 and components of fibrillin-rich microfibrils which form a template for elastin. Fibulin-5 interacted in a dose-dependent manner with a fibrillin-1 N-terminal sequence and with tropoelastin, but not with MAGP-1 (microfibril-associated glycoprotein-1) or decorin. Fibulin-5 did not inhibit interactions between fibrillin-1 N- and C-terminal fragments, or fibrillin-1 interactions with tropoelastin. Fibulin-5 may provide a link between tropoelastin and microfibrils in the pericellular space during elastic fibre assembly.

scholarly journals Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (6) ◽  
pp. 3060-3069 ◽  
Author(s):  
S Irniger ◽  
C M Egli ◽  
G H Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Signal Sequence ◽  
Test System ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sequence Elements ◽  
Polyadenylation Sites ◽  
Point Mutagenesis

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

scholarly journals Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER

1997 ◽  
Vol 110 (8) ◽  
pp. 991-1003 ◽  
Author(s):  
J. Boehm ◽  
F. Letourneur ◽  
W. Ballensiefen ◽  
D. Ossipov ◽  
C. Demolliere ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Retrieval Process ◽  
Hybrid Proteins ◽  
Er Retention ◽  
Golgi Compartment

In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi. In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications. We found that rer3 mutants harbour unique alleles of the alpha-COP-encoding RET1 gene. ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER. Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling. Pulse-chase experiments using these mutants show that alpha-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins. Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner. Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.

Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae

1991 ◽  
Vol 11 (6) ◽  
pp. 3060-3069
Author(s):  
S Irniger ◽  
C M Egli ◽  
G H Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Gene ◽  
Signal Sequence ◽  
Test System ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sequence Elements ◽  
Polyadenylation Sites ◽  
Point Mutagenesis

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

scholarly journals Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast.

1994 ◽  
Vol 127 (3) ◽  
pp. 653-665 ◽  
Author(s):  
E C Gaynor ◽  
S te Heesen ◽  
T R Graham ◽  
M Aebi ◽  
S D Emr
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Minor Role ◽  
Type I ◽  
Dependent Manner ◽  
Coding Sequence ◽  
Golgi Compartment

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.

scholarly journals Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.

1995 ◽  
Vol 131 (4) ◽  
pp. 913-927 ◽  
Author(s):  
M Lussier ◽  
A M Sdicu ◽  
T Ketela ◽  
H Bussey
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Golgi Localization ◽  
Golgi Compartment ◽  
Membrane Spanning

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

Epigenetic Repression of the Adaptor Molecule LAT2 by the Leukemic Fusion Protein AML1/ETO.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 987-987
Author(s):  
Jesus Duque-Afonso ◽  
Tobias Berg ◽  
Olaf Heidenreich ◽  
Michael Luebbert
Keyword(s):  
Fusion Protein ◽  
Molecular Mechanisms ◽  
Hdac Inhibitors ◽  
Histone H3 ◽  
Chimeric Protein ◽  
Dependent Manner ◽  
U937 Cells ◽  
Conditional Expression ◽  
Dose Dependent

Abstract The chromosomal translocation (8;21) fuses the hematopoietic transcription factor AML1 (RUNX1) with ETO, resulting in the leukemia-specific chimeric protein AML1/ETO. This fusion protein represses transcription by recruiting a nuclear co-repressor complex containing HDACs and DNMT1 to its target promoters. Previously, we have identified a novel in vivo AML1/ETO target gene, LAT2 (NTAL/LAB/WBSCR5), which is involved in FcεR I, c-Kit, B cell- and T cell receptor signalling. Notably, LAT2 is strongly repressed in AML1/ETO positive cells including primary AML blasts, which was confirmed by others in several large AML cohorts. We have now addressed the molecular mechanisms of AML1/ETO-mediated LAT2 repression. AML1/ETO was induced by Ponasterone A in an ecdysone-inducible system in U937 cells (9/14/18 cell line). To deplete AML1/ETO in t(8;21)-positive cells, we electroporated Kasumi-1 cells with AML1/ETO siRNA. To interfere with epigenetic modifications more directly, cells were treated with the DNMT inhibitor decitabine (DAC) and 4 different HDAC inhibitors. LAT2 expression was determined by Northern Blot, qRT-PCR and Western Blot. HDAC occupation and the histone status of the LAT2 promoter was examined by chromatin immunoprecipitation (ChIP). LAT2 mRNA was downregulated already after 4 hours of conditional expression of AML1/ETO in 9/14/18 cells, and constitutively repressed in the AML1/ETO-positive Kasumi-1 and SKNO-1 cells. siRNA-mediated AML1/ETO depletion caused a 9-fold upregulation of LAT2 in Kasumi-1 cells, suggesting a possible direct mechanism of repression. To address this question, we performed ChIP assays for the LAT2 promoter after AML1/ETO induction in 9/14/18 cells. AML1/ETO inhibited acetylation of histone H3, H3K9 and H4, but did not affect trimethylation of H3K4. These changes were associated with the recruitment of HDAC2, but not HDAC1 and HDAC3, to the LAT2 promoter. The HDAC inhibitors MS-275, SAHA, TSA and valproic acid induced LAT2 mRNA in a dose-dependent manner in AML1/ETO-expressing Kasumi-1, with MS-275 being the most efficient inhibitor. MS-275 induced LAT2 expression also in t(8;21)-positive SKNO-1, but not in AML1/ETO-negative HL60 and U937 cells. LAT2 mRNA was also upregulated in a dose-dependent manner after DAC treatment in Kasumi-1 cells. The combination of DAC and MS-275 had a synergistic effect on inhibition of cell growth, acetylation of histones H3 and H4, and re-expression of LAT2 mRNA. MS-275-mediated re-expression of LAT2 was associated with an increase in acetylation of histone H3, H3K9, H4 and trimethylation of H3K4. The increase of activating histone modifications was associated with the release of HDAC1, HDAC2 and HDAC3 from the LAT2 promoter. In conclusion, the epigenetic changes of the LAT2 promoter caused by AML1/ETO could be pharmacologically reverted by inhibition of histone acetylation.

scholarly journals A Bni4-Glc7 Phosphatase Complex That Recruits Chitin Synthase to the Site of Bud Emergence

2003 ◽  
Vol 14 (1) ◽  
pp. 26-39 ◽  
Author(s):  
Lukasz Kozubowski ◽  
Heather Panek ◽  
Ashley Rosenthal ◽  
Andrew Bloecher ◽  
Douglas J. DeMarini ◽  
...  
Keyword(s):  
Chitin Synthase ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Emergence ◽  
Bud Neck ◽  
A Cell ◽  
Cycle Dependent

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4V831A/F833A to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4V831A/F833A is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or abni4 V831A/F833A mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.

scholarly journals The C-terminal Region of Mitochondrial Single-subunit RNA Polymerases Contains Species-specific Determinants for Maintenance of Intact Mitochondrial Genomes

2002 ◽  
Vol 13 (7) ◽  
pp. 2245-2255
Author(s):  
Thomas Lisowsky ◽  
Detlef Wilkens ◽  
Torsten Stein ◽  
Boris Hedtke ◽  
Thomas Börner ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Rna Polymerases ◽  
Mitochondrial Genomes ◽  
Mitochondrial Rna ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Stable Maintenance ◽  
Species Specific

Functional conservation of mitochondrial RNA polymerases was investigated in vivo by heterologous complementation studies in yeast. It turned out that neither the full-length mitochondrial RNA polymerase of Arabidopsis thaliana, nor a set of chimeric fusion constructs from plant and yeast RNA polymerases can substitute for the yeast mitochondrial core enzyme Rpo41p when expressed in Δrpo41 yeast mutants. Mitochondria from mutant cells, expressing the heterologous mitochondrial RNA polymerases, were devoid of any mitochondrial genomes. One important exception was observed when the carboxyl-terminal domain of Rpo41p was exchanged with its plant counterpart. Although this fusion protein could not restore respiratory function, stable maintenance of mitochondrial petite genomes (ρ−)− was supported. A carboxyl-terminally truncated Rpo41p exhibited a comparable activity, in spite of the fact that it was found to be transcriptionally inactive. Finally, we tested the carboxyl-terminal domain for complementation intrans. For this purpose the last 377 amino acid residues of yeast mitochondrial Rpo41p were fused to its mitochondrial import sequence. Coexpression of this fusion protein with C-terminally truncated Rpo41p complemented the Δrpo41 defect. These data reveal the importance of the carboxyl-terminal extension of Rpo41p for stable maintenance of intact mitochondrial genomes and for distinct species-specific intramolecular protein–protein interactions.

scholarly journals The WD-repeats of Net2p Interact with Dnm1p and Fis1p to Regulate Division of Mitochondria

2003 ◽  
Vol 14 (10) ◽  
pp. 4126-4139 ◽  
Author(s):  
Kara L. Cerveny ◽  
Robert E. Jensen
Keyword(s):  
Mitochondrial Fission ◽  
Terminal Region ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Dominant Negative Effect ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Wd Repeats

The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae. Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats. We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p. Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p. Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution. Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division. Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division.

Sign in / Sign up

Export Citation Format

Share Document