The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a nonglycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys 48 -specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-␣ and CD3-␦.When a newly synthesized protein molecule is translocated into the ER, 1 there is a fair chance that it may never reach its final destination as a functional molecule, since a significant proportion of newly synthesized proteins is degraded via the endoplasmic reticulum-associated degradation (ERAD) pathway (1). In particular, proteins that misfold along the folding pathway or cannot be appropriately folded as a result of mutations are degraded via this route. The cystic fibrosis transmembrane conductance regulator (CFTR) and its common mutation ⌬F508 in cystic fibrosis serve as an example in this context (2). In addition, proteins that lack their oligomerization partner(s) are prone to degradation, e.g. individual subunits of the T-cell receptor like TCR-␣ and CD3-␦ (3). Finally, ERAD also functions in the homeostatic regulation of metabolic pathways to degrade proteins whose activity needs to be attenuated at a certain metabolic state. Examples include 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) (4), which is further described below, and apolipoprotein B (5).Degradation of proteins from the ER requires dislocation of the substrate from the ER to the cytosol followed by proteolysis via the ubiquitin-proteasome pathway. The dislocation process is thought to require components of the translocon channel, including Sec61␣ (6 -8), as well as a complex of proteins designated CDC48/p97-Ufd1-Npl4 (9 -11). Ubiquitination also plays an essential role in dislocation as illustrated by the inhibition of protein dislocation when the ubiquitination machinery is disrupted (9...
Prokaryotes contain cytoskeletal proteins such as the tubulin-like FtsZ, which forms the Z ring at the cell center for cytokinesis, and the actin-like MreB, which forms a helix along the long axis of the cell and is required for shape maintenance. Using time-lapse analysis of Escherichia coli cells expressing FtsZ-GFP, we found that FtsZ outside of the Z ring also localized in a helix-like pattern and moved very rapidly within this pattern. The movement occurred independently of the presence of Z rings and was most easily detectable in cells lacking Z rings. Moreover, we observed oscillation waves of FtsZ-GFP in the helix-like pattern, particularly in elongated cells, and the period of this oscillation was similar to that of the Min proteins. The MreB helix was not required for the rapid movement of FtsZ or the oscillation of MinD. The results suggest that FtsZ not only forms the Z ring but also is part of a highly dynamic, potentially helical cytoskeleton in bacterial cells.
Formylation of the initiator tRNA is essential for normal growth of Escherichia coli. The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon. However, an additional mutation at position 72 (72A 3 G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation. In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/ U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient. The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA Met in initiation and elongation, in the mitochondria of various organisms.Organisms have evolved with two distinct species of methionyl tRNAs. Of these, the initiator recognizes the initiation codons (AUG, GUG, AUU, UUG, etc.), and the elongator decodes the subsequent AUG codons in a mRNA (1). Both species of the tRNA are aminoacylated by the same methionyl-tRNA synthetase. In eubacteria, the initiators (Met-tRNA fMet ) are then further modified by formyltransferase to generate formylmethionyl-tRNA (fMet-tRNA fMet ), which interacts with IF2 to participate at the step of initiation (2-4). On the other hand, the elongators (Met-tRNA Met ) bind to EFTu to participate in elongation cycles. The major structural features that distinguish the initiators from elongators are located within the acceptor and the anticodon stems (5-7). A striking feature of the eubacterial initiator tRNAs is the presence of a mismatch at the 1-72 positions, which constitutes an important element for their recognition by formyltransferase (8 -11). The mismatch also prevents the binding of the initiators to EF-Tu and renders the fMet-tRNA fMet resistant to peptidyl-tRNA hydrolase, PTH 1 (12), an enzyme which hydrolyzes N-blocked aminoacyl and the peptidyl tRNAs to facilitate tRNA recycling (13,14).The initiator tRNA containing CAU to CUA anticodon change, tRNA fMet (U35A36), is aminoacylated with glutamine (15) and initiates with formyl-glutamine utilizing UAG as an initiation codon in Escherichia coli (16). However, the tRNA fMet (U35A36) was rendered nonfunctional in initiation when coupled with an A to G mutation at position 72 (G72/U35A36). The G72 mutation replaced the CxA mismatch at the top of the acceptor stem with a strong C-G base pair. The G72/U35A36 tRNA could be aminoacylated with glutamine but failed to initiate because it is an extremely poor substrate for formyltransferase (8,17).In this report, we show that a spontaneous intragenic mutation (C 3 T) corresponding ...
The post-transcriptional processing of tRNAs decorates them with a number of modified bases important for their biological functions. Queuosine, found in the tRNAs with GUN anticodons (Asp, Asn, His, Tyr), is an extensively modified base whose biosynthetic pathway is still unclear. In this study, it was observed that the tRNA Tyr from Escherichia coli B105 (a B strain) migrated faster than that from E. coli CA274 (a K-12 strain) on acid urea gels. The organization of tRNA Tyr genes in E. coli B105 was found to be typical of the B strains. Subsequent analysis of tRNA Tyr and tRNA His from several strains of E. coli on acid urea gels, and modified base analysis of tRNA preparations enriched for tRNA Tyr , showed that E. coli B105 lacked queuosine in its tRNAs. However, the lack of queuosine in tRNAs was not a common feature of all E. coli B strains. The tgt and queA genes in B105 were shown to be functional by their ability to complement tgt and queA mutant strains. These observations suggested a block at the step of the biosynthesis of preQ 1 (or preQ 0 ) in the B105 strain. Interestingly, a multicopy vector harbouring a functional tgt gene was toxic to E. coli B105 but not to CA274. Also, in mixed cultures, E. coli B105 was readily competed out by the CA274 strain. The importance of these observations and this novel strain (E. coli B105) in unravelling the mechanism of preQ 1 or preQ 0 biosynthesis is discussed.
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