Sphingolipid metabolism drives mitochondria remodeling during aging and oxidative stress.

One of the hallmarks of aging is a decline in the function of mitochondria, which is often accompanied by altered morphology and dynamics. In some cases, these changes may reflect macromolecular damage to mitochondria that occurs with aging and stress, while in other cases they may be part of a programmed, adaptive response. In this study, we report that mitochondria undergo dramatic morphological changes in chronologically aged yeast cells.

ER-phagy drives age-onset remodeling of endoplasmic reticulum structure-function and lifespan.

The endoplasmic reticulum (ER) comprises an array of structurally distinct subdomains, each with characteristic functions. While altered ER-associated processes are linked to age-onset pathogenesis, whether shifts in ER morphology underlie these functional changes is unclear. We report that ER remodeling is a conserved feature of the aging process in models ranging from yeast to and mammals.

Art2 mediates selective endocytosis of methionine transporters during adaptation to sphingolipid depletion.

Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized might be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor, in Saccharomyces cerevisiae.

Reduced sphingolipid biosynthesis modulates proteostasis networks to enhance longevity.

As the elderly population increases, chronic, age-associated diseases are challenging healthcare systems around the world. Nutrient limitation is well known to slow the aging process and improve health. Regrettably, practicing nutrient restriction to improve health is unachievable for most people.

Use of Deubiquitinase Fusion Proteins to Characterize Endocytic Trafficking in Yeast.

Ubiquitin modification is known to regulate endocytic trafficking of many different types of cargo in eukaryotic cells, but it can be challenging to determine what role, if any, ubiquitin plays in the trafficking of a novel or uncharacterized endocytic cargo. Here, we describe a useful approach that leverages fusion to deubiquitinase (DUB) catalytic domains to explore the role ubiquitin plays in endocytic trafficking. This approach can be applied to the analysis of many different endocytic cargos in different cell types, and it can also be used to study linkage specificity in endocytic trafficking.

Application of Archaea in Deubiquitinase-Like Enzyme Discovery and Activity Assay.

Archaea can be used as microbial platforms to discover new types of deubiquitinase-like (DUB-like) enzymes and to produce ubiquitin/ubiquitin-like (Ub/Ubl) protein conjugates as substrates for DUB/DUB-like activity assays. Here we outline how to use archaea to synthesize, purify, and assay the activity of DUB-like enzymes with unusual properties, including catalytic activity in hypersaline conditions, organic solvents, and high temperatures. We also outline the application of archaea in forming Ub/Ubl isopeptide linkages that include the covalent attachments of diverse archaeal and eukaryotic Ub/Ubls to target proteins.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in .

Regulation of ubiquitin and ubiquitin-like modifiers by phosphorylation.

The regulatory influence of ubiquitin is vast, encompassing all cellular processes, by virtue of its central roles in protein degradation, membrane trafficking, and cell signaling. But how does ubiquitin, a 76 amino acid peptide, carry out such diverse, complex functions in eukaryotic cells? Part of the answer is rooted in the high degree of complexity associated with ubiquitin polymers, which can be 'read' and processed differently depending on topology and cellular context. However, recent evidence indicates that post-translational modifications on ubiquitin itself enhance the complexity of the ubiquitin code.

Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast.

Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood.

Cargo Release from Myosin V Requires the Convergence of Parallel Pathways that Phosphorylate and Ubiquitylate the Cargo Adaptor.

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud.

A Snf1-related nutrient-responsive kinase antagonizes endocytosis in yeast.

Endocytosis is regulated in response to changing environmental conditions to adjust plasma membrane (PM) protein composition for optimal cell growth. Protein networks involved in cargo capture and sorting, membrane sculpting and deformation, and vesicle scission have been well-characterized, but less is known about the networks that sense extracellular cues and relay signals to trigger endocytosis of specific cargo. Hal4 and Hal5 are yeast Snf1-related kinases that were previously reported to regulate nutrient transporter stability by an unknown mechanism.

Rhodanese-Like Domain Protein UbaC and Its Role in Ubiquitin-Like Protein Modification and Sulfur Mobilization in Archaea.

Ubiquitin-like protein (Ubl) modification targets proteins for transient inactivation and/or proteasome-mediated degradation in archaea. Here the rhodanese-like domain (RHD) protein UbaC (HVO_1947) was found to copurify with the E1-like enzyme (UbaA) of the Ubl modification machinery in the archaeon UbaC was shown to be important for Ubl ligation, particularly for the attachment of the Ubl SAMP2/3s to protein targets after exposure to oxidants (NaOCl, dimethyl sulfoxide [DMSO], and methionine sulfoxide [MetO]) and the proteasome inhibitor bortezomib. While UbaC was needed for ligation of the Ubl SAMP1 to MoaE (the large subunit of molybdopterin synthase), it was not important in the formation of oxidant-induced SAMP1 protein conjugates.

Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in .

Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine--sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant.

Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea.

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex.

Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits.

The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1's substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts.

Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea.

Unlabelled: The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes.

Proteome targets of ubiquitin-like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii.

Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known.

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents.

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M.

Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea.

Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase.

Structural and biochemical properties of an extreme 'salt-loving' proteasome activating nucleotidase from the archaeon Haloferax volcanii.

In eukaryotes, the 26S proteasome degrades ubiquitinylated proteins in an ATP-dependent manner. Archaea mediate a form of post-translational modification of proteins termed sampylation that resembles ubiquitinylation. Sampylation was identified in Haloferax volcanii, a moderate halophilic archaeon that synthesizes homologs of 26S proteasome subunits including 20S core particles and regulatory particle triple-A ATPases (Rpt)-like proteasome-associated nucleotidases (PAN-A/1 and PAN-B/2).

Archaeal ubiquitin-like SAMP3 is isopeptide-linked to proteins via a UbaA-dependent mechanism.

SAMP1 and SAMP2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3) with a β-grasp fold and C-terminal diglycine motif characteristic of ubiquitin that is functional in protein conjugation in Hfx. volcanii.

Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin-like small archaeal modifier proteins (SAMPs) from protein-conjugates.

Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H.

E1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur transfer in archaea.

Based on our recent work with Haloferax volcanii, ubiquitin-like (Ubl) proteins (SAMP1 and SAMP2) are known to be covalently attached to proteins in archaea. Here, we investigated the enzymes required for the formation of these Ubl-protein conjugates (SAMPylation) and whether this system is linked to sulfur transfer. Markerless in-frame deletions were generated in H.