Proteomic detection of non-annotated microproteins indicates the translation of hundreds of small open reading frames in human cells, but whether these microproteins are functional is unknown. Here, we report the discovery and characterization of a 7-kilodalton human microprotein we named non-annotated P-body dissociating polypeptide (NoBody). NoBody interacts with mRNA decapping proteins, which remove the 5’ cap from mRNAs to promote 5’-3’ decay. Decapping proteins participate in mRNA turnover and nonsense mediated decay (NMD). NoBody localizes to mRNA decay-associated RNA-protein granules called P-bodies. Modulation of NoBody levels reveals that its abundance is anti-correlated with cellular P-body numbers and alters the steady-state levels of a cellular NMD substrate. These results implicate NoBody as a novel component of the mRNA decapping complex and demonstrate potential functionality of a newly discovered microprotein.

Mycobacterium tuberculosis is the causative agent of the disease tuberculosis (TB). TB kills about 2 million people annually, and approximately one-third of the world's population is currently infected with M. tuberculosis (40). A serious problem in the worldwide fight against TB is the emergence of multidrug-resistant strains of M. tuberculosis. To develop logical targets for new, effective drugs, the physiology of mycobacteria must be better understood.As with all bacterial pathogens, the majority of M. tubercu-

Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36–55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions.

Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. Double-stranded DNA (dsDNA) viruses, such as the tailed phages and herpesviruses, have coat proteins that lack sequence homology but possess a common HK97 fold (1, 2). Herpesviruses infect vertebrate hosts and in humans are responsible for a wide spectrum of diseases causing conditions ranging from cold sores and genital sores to chicken pox and shingles. Thus, understanding the assembly of herpesviruses is crucial for identification of novel drug targets. Bacteriophage P22 is a dsDNA virus with a scaffolding protein-mediated assembly pathway, which is similar to that of herpesvirus. Therefore, P22 provides a good simple model system for studying the capsid assembly of dsDNA viruses (3, 4).In bacteriophage P22, capsid assembly involves interaction of 415 coat protein monomers with ϳ100 molecules of scaffolding protein (5-9). This results in the formation of an intermediate structure called the procapsid (10), into which ejection proteins (11, 12) as well as a portal complex are coassembled (13). DNA gets packaged through the portal complex (5), scaffolding protein exits, and the capsid expands in volume (14). The addition of plug, tail needle, and tailspike proteins results in a mature infectious virion (15).Some coat proteins having the HK97 fold are embellished with extra domains (Fig. 1) (16). P22 coat protein is one of these, as it has a nonconserved accessory domain inserted between the A and P domains (10,17,18). This domain, referred to as the insertion domain (I domain) (19), plays an important role in the folding of coat protein by acting as an intramolecular chaperone (20). The I domain is also involved in determination of capsid size (21) and is hypothesized to stabilize procapsids by forming intersubunit interactions between adjacent capsomers (10,19,20). A recent nuclear magnetic resonance (NMR) solution structure of the isolated I domain revealed a large flexible loop, referred to...

We have discovered the existence of a minor contaminating protein in our preparations of Mycobacterium tuberculosis SecA1 and SecA2 proteins. This minor contaminant is an acetate-stimulated ATPase, which we were able to remove from our protein preparations by the addition of a Q-Sepharose column. The affinity of M. tuberculosis wild-type SecA1, wild-type SecA2, and SecA2 K115R for ATP did not change after the additional purification step. The ATPase activity for M. tuberculosis SecA2 is significantly lower than first reported, and the substitution K115R in SecA2 essentially eliminates the ATPase activity. Nevertheless, the central conclusions of the paper are unchanged.The contaminating ATPase influenced the results of the ATPase assays shown in Fig. 3B and 4C. In the figure below, panel A shows an SDS gel of a representative purification of SecA2 K115R before (lane 1) and after (lane 2) additional purification with the Q-Sepharose column. Panel B shows ATP hydrolysis with time of the SecA2 K115R variant before the Q-Sepharose column in the presence of magnesium acetate (circles) and after the Q-Sepharose column with magnesium acetate (diamonds) and magnesium chloride (squares). The ATPase activity of the proteins was determined by quantifying the amount of inorganic phosphate released from ␥-32 P-labeled ATP with time. These data verify that the acetate-stimulated ATPase was eliminated from our protein. In panel C, the ATP hydrolysis rates of Escherichia coli SecA and Q-Sepharose-purified M. tuberculosis SecA1, SecA2, and SecA2 K115R proteins are shown with 5 mM magnesium chloride used in the reaction buffer. 4051on May 11, 2018 by guest

Background: Mycobacterium tuberculosis possesses a second SecA ATPase that is required for bacterial virulence. Results: ADP binding by SecA2 causes a conformational change observable by biochemical methods. Conclusion: ADP binding closes the clamp in SecA2. This is not observed in SecA1 or E. coli SecA. Significance: Nucleotide-dependent clamp closure suggests a mechanism by which mycobacterial SecA2 is distinguished from SecA1 by the translocation machinery.

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