A novel motif at the C-terminus of palmitoyltransferases is essential for Swf1 and Pfa3 function in vivo

2009 ◽  
Vol 419 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Ayelén González Montoro ◽  
Rodrigo Quiroga ◽  
Hugo J. F. Maccioni ◽  
Javier Valdez Taubas
Keyword(s):  
Membrane Proteins ◽  
Post Translational Modification ◽  
Lipid Molecule ◽  
Additional Information ◽  
C Terminus ◽  
Protein Receptors ◽  
Thioester Bond ◽  
Function Relationship ◽  
Eukaryotic Organisms

S-acylation (commonly known as palmitoylation) is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of a protein through a thioester bond. This modification is predominantly mediated by a family of proteins referred to as PATs (palmitoyltransferases). Most PATs are polytopic membrane proteins, with four to six transmembrane domains, a conserved DHHC motif and variable C-and N-terminal regions, that are probably responsible for conferring localization and substrate specificity. There is very little additional information on the structure–function relationship of PATs. Swf1 and Pfa3 are yeast members of the DHHC family of proteins. Swf1 is responsible for the S-acylation of several transmembrane SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and other integral membrane proteins. Pfa3 is required for the palmitoylation of Vac8, a protein involved in vacuolar fusion. In the present study we describe a novel 16-amino-acid motif present at the cytosolic C-terminus of PATs, that is required for Swf1 and Pfa3 function in vivo. Within this motif, we have identified a single residue in Swf1, Tyr323, as essential for function, and this is correlated with lack of palmitoylation of Tlg1, a SNARE that is a substrate of Swf1. The equivalent mutation in Pfa3 also affects its function. These mutations are the first phenotype-affecting mutations uncovered that do not lie within the DHHC domain, for these or any other PATs. The motif is conserved in 70% of PATs from all eukaryotic organisms analysed, and may have once been present in all PATs. We have named this motif PaCCT (‘Palmitoyltransferase Conserved C-Terminus’).

scholarly journals Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of α-tubulin

2003 ◽  
Vol 375 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Silvia A. PURRO ◽  
C. Gastón BISIG ◽  
María A. CONTIN ◽  
Héctor S. BARRA ◽  
Carlos A. ARCE
Keyword(s):  
Cultured Cells ◽  
In Vivo Studies ◽  
Brain Extract ◽  
Similar Degree ◽  
C6 Cells ◽  
Post Translational Modification ◽  
Α Subunit ◽  
C Terminus

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the α-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of α-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha-ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7–10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of α-tubulin is proposed as one possible cause of reversion of the malignant phenotype.

scholarly journals RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells

2004 ◽  
Vol 24 (13) ◽  
pp. 5746-5756 ◽  
Author(s):  
Michail Shipitsin ◽  
Larry A. Feig
Keyword(s):  
Membrane Proteins ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Amino Acid Sequences ◽  
Exocyst Complex ◽  
C Terminus ◽  
Polarized Delivery ◽  
E Cadherin

ABSTRACT RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.

scholarly journals Lipidation of a bacterial effector is critical for bacterial evasion of host-defense

2020 ◽  
Author(s):  
Natalia Cattelan ◽  
Hongjiao Yu ◽  
Kornelia Przybyszewska ◽  
Rosa Angela Colamarino ◽  
Massimiliano Baldassarre ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Subcellular Fractionation ◽  
Effector Protein ◽  
Host Bacterium ◽  
Post Translational Modification ◽  
C Terminus ◽  
Mouse Macrophages ◽  
Caax Motif ◽  
Shed Light

AbstractThe Rab32 antimicrobial pathway has been shown to restrict Salmonella Typhi, in mouse macrophages. The broad-host pathogen Salmonella Typhimurium however has evolved a strategy to evade the Rab32 antimicrobial pathway, via its effector protein GtgE. GtgE is a cysteine protease that specifically mediates the cleavage and inactivation of Rab32. Here we show that GtgE association and targeting to membranes is critical for its efficient proteolytic activity. The C-terminus of GtgE contains a CaaX motif, which can be post-translationally modified by the host’s prenylation machinery. Using a combination of confocal microscopy and subcellular fractionation we show that a cysteine in the CaaX motif is crucial for GtgE membrane targeting and, more importantly, GtgE localization to the Salmonella-containing vacuole. We also demonstrated that prenylation of CaaX is important for an effective and fast Rab32 cleavage, which in turn helps Salmonella to successfully survive in macrophages and establish an in vivo infection in mice. Our findings shed light on the importance of a host mediated post-translational modification that targets GtgE to the membranes where it can efficiently cleave and inactivate Rab32, leading to a better Salmonella survival in macrophages.Author summarySalmonella species includes a large group of bacteria that cause disease in different hosts. While some serovars are host generalists, others are restricted to humans. This is the case of Salmonella Typhi, responsible for Typhoid fever, a disease that affects millions globally. We have previously discovered an antimicrobial activity in macrophages that is controlled by Rab32. While the broad-host bacterium Salmonella Typhimurium effectively counteracts this mechanism through the delivery of two effectors, GtgE and SopD2, Salmonella Typhi does not express those effectors and cannot survive in mouse macrophages. In this article, we demonstrate how Salmonella Typhimurium exploits a host machinery to modify GtgE. We show that this host mediated modification is important for GtgE intracellular localization and effective Rab32 targeting, resulting in both a better intracellular survival and infection in vivo.

scholarly journals A novel yeast-based high-throughput method for the identification of protein palmitoylation inhibitors

Open Biology ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 200415
Author(s):  
Consuelo Coronel Arrechea ◽  
María Luz Giolito ◽  
Iris Alejandra García ◽  
Gastón Soria ◽  
Javier Valdez Taubas
Keyword(s):  
High Throughput ◽  
Protein S ◽  
Cell Physiology ◽  
Post Translational Modification ◽  
Lipid Molecule ◽  
Protein Palmitoylation ◽  
High Throughput Method ◽  
Thioester Bond ◽  
The Central Nervous System ◽  
Candidate Molecule

Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia . As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.

scholarly journals A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon

2016 ◽  
Vol 113 (39) ◽  
pp. 10902-10907 ◽  
Author(s):  
Emily Breeze ◽  
Natasha Dzimitrowicz ◽  
Verena Kriechbaumer ◽  
Rhiannon Brooks ◽  
Stanley W. Botchway ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Structural Feature ◽  
Mechanism Of Action ◽  
Membrane Curvature ◽  
Amphipathic Helix ◽  
Transmembrane Topology ◽  
C Terminus ◽  
Er Membrane

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

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