Recently, we have localized more than 800 human proteins in living cells with the aim of gaining preliminary functional data [3]. Analysis of these proteins for sequences exhibiting characteristic GTPase motifs such as the P-loop [18] allowed us to identify a subset of proteins as putative GTPases.
Human Lsg1p defines a highly conserved GTPase protein family
One of these proteins possesses a central GTPase domain defined in the PFAM database of protein domains [19] as the MMR/HSR1 domain, a coiled coil, and a potential nuclear localization signal (NLS) (Figure 1A). No additional structural or enzymatic domains could be identified in the protein sequence using the SMART domain research server [20]. Interestingly, this GTPase domain is circularly permuted [21], in contrast to the canonical organization of GTPases based on the small GTPase Ras [12,22]. This circular permutation is unique, and it is surprising in view of the structure of the GTPase P-loop domain [22,23]. It implies that the four highly conserved elements of the GTPase that mediate interactions with the guanine nucleotides and effector proteins, known as the G1, G2, G3 and G4 motifs, are circularly permuted and reorganized, since G4 is followed by G1, G2 and G3 [12,22] (Figure 1A).
A BLAST search for similar protein sequences [15] shows that this unusual GTPase is present as a single copy per genome [22] (Figure 1B [see Additional file 1]). Only one member of the family has so far been experimentally defined, namely the Lsg1 protein in S. cerevisiae [24]. Accordingly, we named the human protein hLsg1 (human orthologue of Lsg1). All orthologues of hLsg1 possess a central MMR/HSR1 domain belonging to KOG1424 in the database of clusters of orthologous genes [25]. The identity between aligned sequences ranges from 31% to 88%. Interestingly, except in the E. cuniculi member, the GTPase domain contains an unusual insertion in comparison to the canonical GTPase structure. This insertion separates the G4 element from the remaining GTPase elements (G1, G2 and G3) (Figure 1A).
In order to elucidate the potential function of hLsg1, we extended our phylogenetic analysis. Owing to their unique structure, circularly permuted GTPases have previously been reported [22,23] and partially grouped into the Yawg/YlqF family (COG1160) [26], which is mainly restricted to prokaryotes and microbial eukaryotes (S. cerevisiae, E. cuniculi). This family contains five subfamilies: YjeQ (YloQ), MJ1464, YqeH, YlqF and Yawg. The latter three branches have eukaryotic members, YlqF representing the ancestor of hLsg1. Interestingly, while the YqeH subfamily is limited to only one member per species (also labeled as Euk-porin in sequence database), and the YjeQ subfamily is mainly restricted to bacteria [22], the YlqF subfamily shows a large expansion of this gene family in eukarya (Figure 1C). The YlqF subfamily can be further subdivided into five clades: YlqF (bacterial), MTG1 (KOG2485), LSG1 (KOG1424), NOG2 (Yawg, KOG2423), and NUG1 (KOG2484) according to the S. cerevisiae nomenclature. The YlqF family expands further in Coelomates [GNL1, 23] and in Deuterostomia (Nucleostemin [27]) (Figure 1B [see Additional file 2]).
Next, we exploited the experimental data from a comprehensive large-scale localization screen in yeast [2] and we conducted literature searches to deduce the possible cellular localizations of the different family members, ranging from the nucleolus to the mitochondria. The nucleolus is the compartment in which the large ribosomal RNA precursor (pre-rRNA) is synthesized, processed into the mature 18S, 5.8S, and 28S rRNAs and assembled with proteins to form ribosomal subunits that move to the nucleoplasm and are finally exported to the cytoplasm. Mitochondria and chloroplasts also possess a set of ribosomes. All yeast members (LSG1, NOG2, NUG1 and MTG1) are involved in ribosome biogenesis [24,28-30], and YjeQ binds to the ribosome in E. coli [31]. Finally, using ChloroP [32] to predict proteins localized to the chloroplast, we detected a sixth subfamily in YlqF, called ChYlqF (for Chloroplast YlqF), and a second subfamily in YqeH, called ChYqeH (for chloroplast YqeH). These are only found in plant genomes and group in the phylogenetic tree with the cyanobacteria YRG and YqeH members (Figure 1B [see Additional files 1 and 2]).
Nucleotide binding and GTPase activity of hLsg1
Lsg1-related proteins contain motifs that have been found to be important for guanine nucleotide binding and GTPase activity in a variety of cellular proteins [33]. Lsg1-related proteins contain the G1-4 motifs typical of GTPases (Figure 1A), suggesting that members of this family are likely to exhibit guanine nucleotide binding and GTPase activity. However, direct experimental evidence for this has been lacking so far, except for the distantly related bacterial homologues YjeQ [31], YlqF and YqeH [34]. To test this function in human Lsg1, we examined the binding of [32P] GTP to purified His tagged-hLsg1 using as control a His-tagged Sar1p, a well characterized GTPase regulating the vesicular coat complex COPII [35,36]. As shown in Figure 2A, hLsg1 binds to [32P] GTP, although more weakly than Sar1p. However, hLsg1 did not bind GDP under those experimental conditions (data not shown), which may reflect weak binding. To determine the GTPase activity of hLsg1, we performed a GTPase assay using an HPLC system as previously described [17]. In this assay, purified recombinant hLsg1 showed a low GTPase activity that proceeded to GMP and induced the further hydrolysis of GDP through GMP to guanosine. Such low GTPase activities have previously been observed in the distantly related bacterial homolog YjeQ [21], but also in GTPases in general, since their activities rely heavily on co-factors such as GAPs (GTPase Activating Proteins) [41] or GEFs (Guanine nucleotide Exchange Factors) [37]. Moreover, other GTPases such as the interferon-induced 67-kDa guanylate-binding protein (hGBP1) have been shown not to limit their hydrolysis to GDP [42]. To confirm our observations indicating that hLsg1 has GTPase activity, we immunoprecipitated endogenous hLsg1 from a HeLa cell extract using a polyclonal antibody raised against purified hLsg1, and analyzed the GTPase activity of the precipitate (Figure 2B). The GTPase activity was four times higher (incubation time 4 h compared to 18 h required for completion of GTP hydrolysis) than that of in vitro purified recombinant hLsg1 and GDP was the only final product (Figure 2B, lower panel). These data demonstrate GTPase activity in a eukaryotic member of the YlqF family for the first time.
hLsg1 is an essential protein, a characteristic of YRG family members
Yeast Lsg1, like the yeast YRG homologues NUG1, NOG2 and MTG1, is an essential protein [4]. To confirm the consequences of loss of hLsg1, we transfected siRNAs targeted against hLsg1 into HeLa cells, confirming the efficiency of the siRNA treatment by western blot analysis (Figure 3A). After 24 h, Lsg1 expression showed a drastic decrease in cells treated with Lsg1 siRNA compared to cells treated with a negative control siRNA. Moreover, hLsg1 expression in control cells or cells transfected with the negative control shows a band shift that increases with time, as observed in proteins post-translationally modified e.g. by phosphorylation. The increase in intensity could indicate that the polyclonal antibody has a higher affinity for the modified form. There was no significant change in actin expression in control cells, or in cells treated with either random siRNA or a specific hLsg1 siRNA (Figure 3A). However, microscopic observations during the course of the experiment showed that HeLa cell cultures exhibiting hLsg1 knockdown were less dense than control cells. In addition, hLsg1 knockdowns contained more apoptotic cells (not shown), suggesting a lethal effect. We confirmed this by immunostaining hLsg1-specific siRNA-treated cells with a polyclonal anti-hLsg1 antibody and staining the cell nuclei with DAPI at different times after transfection of the siRNA (Figure 3B). Cell numbers decreased rapidly after treatment with the specific hLsg1 siRNA in comparison to cells treated with oligofectamine alone or with control siRNA.
We used the large datasets from gene viability screens of bacteria, worms and flies to compare our observations with data about other YRG family members. YjeQ was shown to be indispensable for the growth of E. coli and B. subtilis [38]. In C. elegans, YRG orthologues are non-viable (t19a6.2a, t19a6.2b, k01c8.9, C53H9) (Figure 3C). Since large human RNAi screens are only now in progress, no data were available for other YRG human genes. However, interestingly, overexpression of nucleostemin was shown to be lethal [27].
According to our results, hLsg1 is essential, like its yeast counterpart, and this characteristic seems to be common to the YRG family members. This implies that each YRG protein fulfils essential functions.
hLsg1 localizes to the endoplasmic reticulum and to discrete nuclear structures
Compartmentalization of the human cell allows better control of function and reactions steps in many pathways, including ribosome assembly. Cellular localization is a key to defining protein function. Using large-scale localization screens, we previously identified hLsg1 as an endoplasmic reticulum localized protein [3], in contrast to yeast Lsg1, which is proposed to localize specifically to the cytosol [24].
We decided to confirm our preliminary data on hLsg1 localization in humans using GFP-fused constructs as well as specific polyclonal antibodies. When expressed as a C-terminally tagged YFP fusion protein, hLsg1 localized to the ER in most cells (Figure 4A, 1). In 10% of the transfected cells, however, discrete structures in the nucleus were observed and localization to the endoplasmic reticulum was decreased or even absent (Figure 4A, 2). An N-terminally tagged CFP-hLsg1 fusion protein was also localized to the ER and nuclear envelope, but more of the protein was cytosolic than in the case of the C-terminal hLsg1-YFP fusion (Figure 4A, 3). A truncated hLsg1 version (480 to 658aa) fused to the YFP, containing the potential NLS, accumulated in the nucleus and nucleolus (Figure 4A, 4). Collectively, these data indicate that the NLS present in the C-terminus of hLsg1 is functional, in contrast to the putative NLS in yeast Lsg1, which is reportedly restricted to the cytosol [24].
Immunostaining with an antibody against the entire protein showed that the endogenous protein also localized to reticular membranes, and in a fraction of the cells to a number of small punctuate nuclear structures. These results are very similar to those obtained with the hLsg1-YFP fusion protein (Figure 4A, 2). Double staining showed that hLsg1 partially co-localized with an ectopically expressed FP (fluorescent protein) used to mark the ER (Clontech ER marker) (Figure 4B, bottom row), as well as with the nuclear envelope marker lamin B1 (Figure 4B, top row). However, hLsg1 was largely absent from the Golgi complex, which was labeled with antibodies against the Golgi membrane protein golgin97, and from mitochondria, marked by antibodies against HSP60 (data not shown). Moreover, the small hLsg1-positive nuclear structures observed in a fraction of the cells co-localized with coilin, a typical marker of Cajal bodies (CBs) (indicated by arrowheads in Figure 4B, middle row). The CBs are functionally linked to the nucleolus and play a major role in the maturation of RNP, acting on the mRNA as well as the rRNA pathway [44].
These data demonstrate that in contrast to its yeast counterpart, hLsg1 localizes to the ER and to Cajal bodies in the nucleus.
hLsg1 shuttles between the nucleus and the cytosol
The dual localization of hLsg1 in the cytosol and nucleus suggests nucleocytoplasmic trafficking of the protein, possibly in relation to rRNA maturation. We constructed hLsg1 deletion mutants containing or excluding the putative the C-terminal NLS (YFP-hLsg1-1-600 and YFP-hLsg1-480-658) and transfected them into Hela cells (Figure 5A). While YFP-hLsg1-480-658 clearly localized in the nucleus, YFP-hLsg1-1-600, which contains no NLS, was excluded from the nucleus. Moreover, YFP-hLsg1-480-658 colocalized in the nucleus with SRP19-MRFP, a nucleolar marker [46], indicating that it sublocalizes to the nucleolus.
To determine whether hLsg1 shuttled between nucleus and cytosol via a CRM1-dependent nuclear export pathway, we transfected Vero cells with either hLsg1-YFP or hLsg1 deletion mutants and compared the localization of the fusion proteins after treatment with the CRM-1 nuclear export inhibitor Leptomycin B (LMB) (Figure 5B). Full-length hLsg1 (YFP-hLsg1) is LMB-sensitive (Figure 5B); so is its C-terminal counterpart hLsg1-CFP (data not shown). To confirm this, we performed the same experiment using the deletion mutants YFP-hLsg1-1-600 and YFP-hLsg1-480-658 as well as the full length YFP-hLsg1. We also took intermediate time points (3 h and 5 h) to obtain insights into the kinetics of hLsg1 shuttling. Interestingly, YFP-hLsg1 accumulates in the nucleus over an 8 h period, and at 5 h most of the transfected cells showed punctate labeling in the nucleus reminiscent of Cajal bodies. YFP-hLsg1-480-658 showed a permanent nuclear location and YFP-hLsg1-1-600 was constantly in the cytosol.
These data suggest that hLsg1 shuttles between the cytosol and Cajal bodies via a CRM1-dependent export mechanism.
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