scholarly journals Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (11) ◽  
pp. 5021-5037 ◽  
Author(s):  
Mark Trautwein ◽  
Jörn Dengjel ◽  
Markus Schirle ◽  
Anne Spang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Short Range ◽  
Small Gtpase ◽  
Polya Tail ◽  
Cellular Processes ◽  
Golgi Membranes ◽  
The Stability ◽  
Ash1 Mrna

The small GTPase Arf1p is involved in different cellular processes that require its accumulation at specific cellular locations. The recruitment of Arf1p to distinct points of action might be achieved by association of Arf1p with different proteins. To identify new interactors of Arf1p, we performed an affinity chromatography with GTP- or GDP-bound Arf1p proteins. A new interactor of Arf1p-GTP was identified as Pab1p, which binds to the polyA-tail of mRNAs. Pab1p was found to associate with purified COPI-coated vesicles generated from Golgi membranes in vitro. The stability of the Pab1p–Arf1p complex depends on the presence of mRNA. Both symmetrically distributed mRNAs as well as the asymmetrically localized ASH1 mRNA are found in association with Arf1p. Remarkably, Arf1p and Pab1p are both required to restrict ASH1 mRNA to the bud tip. Arf1p and coatomer play an unexpected role in localizing mRNA independent and downstream of the SHE machinery. Hereby acts the SHE machinery in long-range mRNA transport, whereas COPI vesicles could act as short-range and localization vehicles. The endoplasmic reticulum (ER)–Golgi shuttle might be involved in concentrating mRNA at the ER.

scholarly journals New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 384
Author(s):  
Myriam Salameh ◽  
Sylvie Riquier ◽  
Olivier Guittet ◽  
Meng-Er Huang ◽  
Laurence Vernis ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Expression Profiles ◽  
Mechanisms Of Action ◽  
Cytosolic Ph ◽  
Cytosolic Domains ◽  
The Stability ◽  
Distinct Features ◽  
Ph Variations ◽  
Biochemical Features

Human CISD2 and mitoNEET are two NEET proteins anchored in the endoplasmic reticulum and mitochondria membranes respectively, with an Fe–S containing domain stretching out in the cytosol. Their cytosolic domains are close in sequence and structure. In the present study, combining cellular and biochemical approaches, we compared both proteins in order to possibly identify specific roles and mechanisms of action in the cell. We show that both proteins exhibit a high intrinsic stability and a sensitivity of their cluster to oxygen. In contrast, they differ in according to expression profiles in tissues and intracellular half-life. The stability of their Fe–S cluster and its ability to be transferred in vitro are affected differently by pH variations in a physiological and pathological range for cytosolic pH. Finally, we question a possible role for CISD2 in cellular Fe–S cluster trafficking. In conclusion, our work highlights unexpected major differences in the cellular and biochemical features between these two structurally close NEET proteins.

scholarly journals RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport

2000 ◽  
Vol 11 (9) ◽  
pp. 3155-3168 ◽  
Author(s):  
Brandon M. Sullivan ◽  
Kimberly J. Harrison-Lavoie ◽  
Vladimir Marshansky ◽  
Herbert Y. Lin ◽  
John H. Kehrl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Rgs Proteins ◽  
Placental Alkaline Phosphatase ◽  
Protein Signaling ◽  
Golgi Membranes

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.

scholarly journals Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Vol 113 (28) ◽  
pp. E4015-E4024 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Disulfide Isomerase ◽  
Misfolded Proteins ◽  
Disulfide Isomerase ◽  
Licensing Factor ◽  
Folded Proteins ◽  
Protein Disulfide

Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p–Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae. We reconstitute this step in vitro with Htm1p–Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p–Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p–Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

scholarly journals The Sar1 Gtpase Coordinates Biosynthetic Cargo Selection with Endoplasmic Reticulum Export Site Assembly

2001 ◽  
Vol 152 (1) ◽  
pp. 213-230 ◽  
Author(s):  
Meir Aridor ◽  
Kenneth N. Fish ◽  
Sergei Bannykh ◽  
Jacques Weissman ◽  
Theresa H. Roberts ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Small Gtpase ◽  
Live Cells ◽  
Tubular Structures ◽  
Golgi Transport ◽  
Er Export

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

scholarly journals Structure–function insights into direct lipid transfer between membranes by Mmm1–Mdm12 of ERMES

2017 ◽  
Vol 217 (3) ◽  
pp. 959-974 ◽  
Author(s):  
Shin Kawano ◽  
Yasushi Tamura ◽  
Rieko Kojima ◽  
Siqin Bala ◽  
Eri Asai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Kluyveromyces Lactis ◽  
Lipid Transfer ◽  
Mitochondrial Membranes ◽  
Hydrophobic Pocket ◽  
X Ray ◽  
Lipid Exchange ◽  
Complex Functions

The endoplasmic reticulum (ER)–mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12–Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1–Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes.

scholarly journals Volumetric Properties of Four-Stranded DNA Structures

Biology ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 813
Author(s):  
Tigran V. Chalikian ◽  
Robert B. Macgregor
Keyword(s):  
Regulation Of Transcription ◽  
Solvent Interactions ◽  
Dna Structures ◽  
Cellular Processes ◽  
Molecular Determinants ◽  
Telomere Biology ◽  
The Stability

Four-stranded non-canonical DNA structures including G-quadruplexes and i-motifs have been found in the genome and are thought to be involved in regulation of biological function. These structures have been implicated in telomere biology, genomic instability, and regulation of transcription and translation events. To gain an understanding of the molecular determinants underlying the biological role of four-stranded DNA structures, their biophysical properties have been extensively studied. The limited libraries on volume, expansibility, and compressibility accumulated to date have begun to provide insights into the molecular origins of helix-to-coil and helix-to-helix conformational transitions involving four-stranded DNA structures. In this article, we review the recent progress in volumetric investigations of G-quadruplexes and i-motifs, emphasizing how such data can be used to characterize intra-and intermolecular interactions, including solvation. We describe how volumetric data can be interpreted at the molecular level to yield a better understanding of the role that solute–solvent interactions play in modulating the stability and recognition events of nucleic acids. Taken together, volumetric studies facilitate unveiling the molecular determinants of biological events involving biopolymers, including G-quadruplexes and i-motifs, by providing one more piece to the thermodynamic puzzle describing the energetics of cellular processes in vitro and, by extension, in vivo.

scholarly journals Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

scholarly journals RNase/Anti-RNase Activities of the Bacterial parD Toxin-Antitoxin System

2005 ◽  
Vol 187 (9) ◽  
pp. 3151-3157 ◽  
Author(s):  
Ana J. Muñoz-Gómez ◽  
Marc Lemonnier ◽  
Sandra Santos-Sierra ◽  
Alfredo Berzal-Herranz ◽  
Ramón Díaz-Orejas
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Rna Degradation ◽  
Double Stranded Rna ◽  
Cellular Processes ◽  
Inhibition Of Protein Synthesis ◽  
Reticulocyte Lysates ◽  
The Stability ◽  
Cole1 Replication

ABSTRACT The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single- and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

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