The role of MeH73 in actin polymerization and ATP hydrolysis 1 1Edited by R. Huber

114

We have shown that Rpl3, a protein of the large ribosomal subunit from baker's yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by topdown and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-L-[methyl-3 H]methionine. The results show that a ؉14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven ␤-strand methyltransferase, results in the loss of the ؉14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.The addition of methyl groups to proteins from the methyl donor S-adenosylmethionine is one of the most common posttranslational modifications, resulting in an expansion of the physico-chemical characteristics of amino acids and the potential to modulate protein function (1). Major sites of protein methylation are at lysine and arginine residues (2, 3), and less major sites include glutamate, glutamine, and histidine residues, as well as N-terminal amino and C-terminal carboxyl groups (4 -6). The extensive role of histone methylation in transcriptional control highlights the biological significance of this modification (7-10). Protein methylation is also important in the translational machinery. Indeed, many proteins involved in translation, including ribosomal proteins and various elongation and release factors, are subject to methylation in both prokaryotes and eukaryotes (11).Saccharomyces cerevisiae is an ideal organism to investigate the methylation of ribosomal proteins; its genome is well annotated and single open reading frame gene deletion mutants are available. High-resolution intact mass spectrometry suggested that six proteins of the large ribosomal subunit may be methylated: Rpl1ab, Rpl3, Rpl12ab, Rpl23ab, Rpl42ab, and Rpl43ab (12).2 This study, however, did not identify the sites of methylation in these proteins nor did it identify the corresponding methyltransferases. In our laboratory, we have been interested in characterizing these modifications and identifying the methyltransferases involved in an effort to understand their physiological significance in trans...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

Webb

Zurita-Lopez

Al-Hadid

et al. 2010

114

“…His 73 in the sensory loop is methylated in muscle actin but not in yeast or either of the two hybrids. This histidine residue has been suggested to play a role in establishing the stability of the filament due to its potential ability to retard the release of organic phosphate generated after ATP hydrolysis (39,40). The difference in exchange observed with peptide 68 -84 for hybrid actins could be partly due to the lack of methylation on His 73 .…”

Section: Discussionmentioning

confidence: 99%

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G-and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1-3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with ␤-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could ex...

“…Recent reports have questioned the validity of the structure of TMR-modified actin with bound ADP as truly representative of the ADP state (17)(18)(19). Some (18,20,21) think that in the "real" ADP state the cleft that separates the two major domains of actin must be open, as observed in the so-called "open" state structure of the actinprofilin complex determined with bound ATP (22).…”

mentioning

confidence: 99%

Crystal Structure of Monomeric Actin in the ATP State

Graceffa

Domínguez

2003

215

110

A nucleotide-dependent conformational change regulates actin filament dynamics. Yet, the structural basis of this mechanism remains controversial. The x-ray crystal structure of tetramethylrhodamine-5-maleimide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-Å resolution. A comparison of this structure to that of tetramethylrhodamine-5-maleimide-actin with bound ADP, determined previously under similar conditions, reveals how the release of the nucleotide ␥-phosphate sets in motion a sequence of events leading to a conformational change in subdo- The actin filament (F-actin) is asymmetric, undergoing net incorporation of ATP-actin monomers to the barbed end and dissociation of ADP-actin monomers from the pointed end (1, 2). This dynamic process, known as actin filament treadmilling, is an essential part of many forms of cell motility. A driving force behind actin treadmilling is the hydrolysis of ATP by actin. However, in vivo treadmilling is further regulated by a number of factors including a battery of actin-binding proteins. Actin-depolymerizing factor/cofilin, for instance, binds preferentially to the ADP-actin monomers that accumulate toward the pointed end of the filament, accelerating their dissociation. Other proteins such as profilin and thymosin-␤4 bind ATPactin with higher affinity than ADP-actin, maintaining a pool of ATP-actin monomers ready for incorporation into the barbed end of the filament. The fact that these proteins can "distinguish" between ATP-and ADP-actin suggests that these two states are structurally different. Consistent with this view, biochemical (3-5), spectroscopic (6 -8), and electron microscopic (9) evidence has suggested that a conformational change in actin subdomain 2 accompanies the hydrolysis of ATP and the release of inorganic phosphate.