Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

Mansilla, Francisco; Friis, Irene; Jadidi, Majid; Nielsen, Karen Margrethe; Clark, Brian F. C.; Knudsen, Charlotte R.

doi:10.1042/bj20011681

Cited by 50 publications

(66 citation statements)

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“…Interestingly, ectopic expression of eEF1A2 in these proliferating CHO cells resulted in considerably more of this protein in the GDP-associated form than observed when eEF1A1 was overexpressed in the same cells (Figure 3c). These findings are consistent with previous studies showing that, compared with eEF1A1, eEF1A2 has a higher preference for binding GDP than GTP in vitro (Kahns et al, 1998), and that it has little or no affinity for the guanine exchange factor, eEF1B, which facilitates the exchange of GDP for GTP on eEF1A1 (Mansilla et al, 2002). Together, these findings indicate that, similar to eEF1A1, the GDP-bound form of eEF1A2 seems to be responsible for the effect of this protein on SK1.…”

Section: Resultssupporting

confidence: 82%

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis

2010

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Sphingosine kinase 1 (SK1) catalyses the formation of bioactive phospholipid sphingosine 1-phosphate (S1P). Elevated cellular SK1 activity and S1P levels enhance cell proliferation and survival, and are strongly implicated in tumourigenesis. Regulation of SK1 activity can occur through various mechanisms, including phosphorylation and protein-protein interactions. We have previously shown that eukaryotic elongation factor 1A (eEF1A) interacts with and directly activates SK1, but the mechanisms regulating this were undefined. Notably, eEF1A has GTPase activity and can exist in GTP-or GDP-bound forms, which are associated with distinct structural conformations of the protein. Here, we show that the guanine nucleotide-bound state of eEF1A regulates its ability to activate SK1, with eEF1A.GDP, but not eEF1A.GTP, enhancing SK1 activity in vitro. Furthermore, we show that enhancing cellular eEF1A. GDP levels through expression of a guanine nucleotide dissociation inhibitor of eEF1A, translationally controlled tumour protein (TCTP), increased SK1 activity in cells. We also examined a truncated isoform of eEF1A1, termed prostate tumour inducer-1 (PTI-1), which can induce neoplastic cell transformation through undefined mechanisms. PTI-1 lacks the G protein domain of eEF1A1 and is therefore unable to undergo the GTP-binding-induced conformational change. Notably, we found that PTI-1 can directly activate SK1 and that this seems to be essential for neoplastic transformation induced by PTI-1, as chemical SK1 inhibitors or overexpression of a dominant-negative SK1 blocked this process. Thus, this study defines the mechanism regulating eEF1A-mediated SK1 activation, and also establishes SK1 as being integral for PTI-1-induced oncogenesis.

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Section: Resultssupporting

confidence: 82%

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis

2010

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“…Two subunits of the eEF1 complex were detected, eEF1A and eEF1G, in addition to eukaryotic translation elongation factor 2 (eEF2) and the α-subunit of the eukaryotic translation initiation factor 3 (eIF3A). The nonribosomal eEF1 protein complex composed of eEF1A, eEF1G, eEF1B2 (also called, eEF1Bα), eEF1D (also called eEF1δ or eEF1Bβ), and valyltRNA synthetase is required for eukaryotic translation (10) (Fig. S2A).…”

Section: Purification and Identification Of Cellular Proteins In Fracmentioning

confidence: 99%

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Warren

Wei

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus. purification | mass spectrometry | cellular factor | siRNA knock down | virus replication T he HIV type-1 (HIV-1) reverse transcriptase (RT) enzyme carries out reverse transcription through DNA polymerase and RNase H activities, which mediate a sequential series of steps that include the initiation of DNA synthesis producing negative strand strong stop DNA (−ssDNA), full-length negative-strand DNA, positive-strand DNA, and intact preintegrative doublestrand DNA (reviewed elsewhere 1). The ability of HIV-1 to efficiently produce early reverse transcription products in vitro has been described (2). However, under in vitro conditions, the production of late reverse transcription products is less efficient than that observed in HIV-1-infected cells (3, 4), suggesting that host cellular factors are required. After entry into the host cell cytoplasm, the HIV-1 core, containing the viral genomic RNA, RT, and integrase (IN), reorganizes to form the reverse transcription complex (RTC). The RTC requires both IN and RT to be active (5), and they are believed to recruit cellular factors to facilitate DNA synthesis (6). Although two-hybrid library and genomewide RNAi screening methods have implicated more than 50 cellular proteins as important for reverse transcription (6, 7), whether any of these cellular proteins are RTC components, or whether they direct the RTC in other ways, is unclear.Attempts to purify and to identify cellular RTC cofactors by more conventional methods ...

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“…A complex containing the four subunits of elongation factor 1 (EF1A and the guanine nucleotide exchange factors EF1B␣, EF1B␤, and EF1B␥) and ValRS (valyl-tRNA synthetase) was described (2, 3). Several structural models of the ValRS-EF1A-guanine nucleotide exchange factor assembly have been proposed (2,4,5). This complex is believed to play a role in channeling of tRNA Val during translation (6).…”

mentioning

confidence: 99%

Dissection of the Structural Organization of the Aminoacyl-tRNA Synthetase Complex

Kamińska

Havrylenko

Decottignies

et al. 2009

Journal of Biological Chemistry

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The spatio-temporal organization of proteins within the cytoplasm of eukaryotic cells rests in part on the assembly of stable and transient multiprotein complexes. Here we examined the assembly of the multiaminoacyl-tRNA synthetase complex (MARS) in human cells. This complex contains nine aminoacyltRNA synthetases and three auxiliary proteins and is a hallmark of metazoan species. Isolation of the complexes has been performed by tandem affinity purification from human cells in culture. To understand the rules of assembly of this particle, expression of the three nonsynthetase components of MARS, p18, p38, and p43, was blocked by stable small interfering RNA silencing. The lack of these components was not lethal for the cells, but cell growth was slightly reduced. The residual complexes that could form in vivo in the absence of the auxiliary proteins were isolated by tandem affinity purification. From the repertoire of the subcomplexes that could be isolated, a comprehensive map of protein-protein interactions mediating complex assembly is deduced. The data are consistent with a structural role of the three nonsynthetase components of MARS, with p38 connecting two subcomplexes that may form in the absence of p38.Multiprotein complexes are molecular machines that are essential for organization of the proteome and for integration of cellular functions. Translation of genetic information involves several supramolecular assemblies, including the ribosome and multiprotein complexes involved in the initiation and elongation steps of the protein biosynthesis process (1). A complex containing the four subunits of elongation factor 1 (EF1A and the guanine nucleotide exchange factors EF1B␣, EF1B␤, and EF1B␥) and ValRS (valyl-tRNA synthetase) was described (2, 3). Several structural models of the ValRS-EF1A-guanine nucleotide exchange factor assembly have been proposed (2,4,5). This complex is believed to play a role in channeling of tRNA Val during translation (6). A multiaminoacyl-tRNA synthetase complex (MARS) 4 of about 1.5 MDa was described more than 20 years ago, but its physical and functional organization remain elusive (7). This complex is ubiquitous from Drosophila to mammals and contains the nine aminoacyl-tRNA synthetases ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, ProRS, and the three nonsynthetase components p18, p38, and p43 (8). Initial protein-protein interaction maps have been determined by using the yeast two-hybrid system (9, 10) or by in vitro cross-linking (11). A structural working model has been proposed by electron microscopy and three-dimensional reconstruction (12). The p38 component forms the platform for complex assembly (9, 13). A p38 deficiency is lethal in mice (14). Recent studies have indicated that some of the components of this complex lead a double life. They are essential components of translation when associated within MARS but may also play noncanonical functions after dissociation from the complex or following transport in other cellular compartments. LysRS associates wit...

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Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

Cited by 50 publications

References 50 publications

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Dissection of the Structural Organization of the Aminoacyl-tRNA Synthetase Complex

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