RESEARCH PROJECTS

POST-TRANSLATIONAL MODIFICATION IN ARCHAEA

One of the first hurdles to be negotiated in the post-genomic era involves the description of the entire protein content of the cell, the proteome. Such efforts are presently complicated by the various post-translational modifications proteins can experience, including glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation and proteolytic cleavage. Whereas these and other post-translational protein modifications have been well-characterized in Eukarya and Bacteria, post-translational modification in Archaea has received far less attention. Although archaeal proteins can undergo post-translational modifications reminiscent of what their eukaryal and/or bacterial counterparts experience, examination of archaeal post-translational modification often reveals aspects of post-translational modification not previously observed in the other two domains of life. In some cases, post-translational modification allows a protein to survive the extremophilic conditions often encountered by Archaea. Various post-translational modifications experienced by archaeal proteins, the molecular steps leading to these modifications as well as the role played by post-translational modification in Archaea are currently being investigated.

Signal peptide cleavage

Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features alongside traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile and oligomeric status. We have recently described genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii (Fine et al., 2006). Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. Employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea like Haloferax volcanii, the signal peptide-cleaving activity of both isolated membranes and purified Sec11a and Sec11b was addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions.

While tending to share little overall resemblance, SPases in Eukarya and Bacteria include five regions of significant sequence homology, termed boxes A-E, with boxes B-E participating in the catalytic cycle of the enzyme. Nonetheless, bacterial and eukaryal SPases differ enzymatically and structurally. In the bacterial enzyme, the box B region contains the conserved nucleophilic Ser-90 (Escherichia coli numbering) residue, while the proposed general base Lys-145 is found in box D. Ser-90 and Lys-145 are believed to form the catalytic dyad responsible for the proteolytic action of the enzyme. By contrast, eukaryal type I SPases have replaced the essential lysine of the bacterial catalytic dyad with a histidine residue. Thus, while their catalytic mechanism remains to be elucidated, eukaryal SPases may rely on either a Ser-His dyad or a Ser-His-Asp triad for catalytic activity rather than the Ser-Lys dyad employed by the bacterial enzyme. The bacterial and eukaryal enzymes also differ in terms of their oligomeric status. Unlike the bacterial enzyme, which functions independently, i.e. as a single encoded polypeptide, eukaryal SPases function as part of a multisubunit SPase complex (SPC).

Whereas type I SPases are relatively well-described in Bacteria and Eukarya, little is known of signal peptide cleavage in Archaea. While containing the evolutionarily-conserved regions of sequence homology, archaeal SPases lack the conserved lysine of the bacterial Ser-Lys catalytic dyad, and, like Eukarya, contain a histidine residue at this position. Thus, archaeal SPases may rely on a catalytic mechanism similar to that used by the eukaryal enzyme. The inability of genomic searches to thus far detect Eukarya-like SPase complex subunits in Archaea suggests that the archaeal enzyme operates independently, as in Bacteria. Furthermore, like bacterial SPases, certain archaeal enzymes include a stretch of residues not found in eukaryal SPases that comprises domain II, a structural motif of unknown function. Thus, current understanding suggests that archaeal SPase may represent an evolutionary intermediate between present-day eukaryal and bacterial enzymes.

Both Sec11a and Sec11b are expressed in H. volcanii but only Sec11b is essential. A. RNA was extracted from H. volcanii and used to direct the synthesis of cDNA. PCR was then performed in the absence of nucleic acids (- ; lane 1), or in the presence of genomic DNA (+DNA; lane 2), total RNA (+RNA; lane 3) or cDNA (+cDNA; lane 4) using primers against either sec11a (left panel) or sec11b (right panel). B. PCR amplification of H. volcanii cells transformed with plasmids pIDT-Sec11a or pIDT-Sec11b, designed to delete either sec11a or sec11b, respectively. The arrows show the positions of the PCR amplification product encoding the Sec11a or Sec11b flanking regions separated by the H. volcanii trpA gene, presented in the last lane of each panel. Also shown in each panel are PCR products using genomic DNA (lane 1), genomic DNA including the integrated pIDT-Sec11a or pIDT-Sec11b plasmids (lane 2), and the same DNA taken from cells grown in tryptophan-free, 5-FOA- and uracil-containing medium, i.e. following expulsion of the plasmid and native sec11a- or sec11b-encoding gene (lane 3), as template. In the right panel, lane 4 shows the PCR product of the cells considered in lane 3 but returned to tryptophan-containing medium such that DNA encoding the sec11a or sec11b flanking regions separated by the H. volcanii trpA gene is expelled from the genome with the plasmid. C. RNA was extracted from H. volcanii cells from which the Sec11a-encoding gene had been deleted and used to direct the synthesis of cDNA. PCR was then performed in the absence of nucleic acids (- ; lane 1), or in the presence of genomic DNA taken from untreated cells (+DNA; lane 2), or total RNA (+RNA; lane 3) or cDNA (+cDNA; lane 4) from the deletion strain, using primers against either sec11a (left panel) or sec11b (right panel).

Purified CBD-Sec11a and CBD-Sec11b are active. A. CBD-Sec11a and B. CBD-Sec11b were purified and tested for their abilities to release the signal peptide (SP) domain from the reporter substrate SP-CBD. Incubations were conducted in the presence of purified enzyme and substrate, enzyme alone or substrate alone, from 0 to 60 minutes.

N-glycosylation in Archaea

A major project in the lab addresses characterization of the N-glycosylation process in Haloferax volcanii. Initially (Abu Qarn and Eichler, 2006), putative H. volcanii homologues of genes involved in eukaryal or bacterial N-glycosylation were identified by bioinformatics. RT-PCR confirmed that the proposed N-glycosylation genes are transcribed, indicative of true proteins being encoded. Where families of related gene sequences were detected, differential transcription of family members under a variety of physiological and environmental conditions was shown. Gene deletions point to certain genes as being essential yet revealed that others are not. Thus, with genes putatively involved in H. volcanii protein glycosylation identified and the ability to address the roles played by the encoded polypeptides in modifying a reporter glycoprotein, the steps of the archaeal N-glycosylation pathway can be defined.

Gene deletion shows that alg5-A is essential for H. volcanii survival. (A) PCR was performed using primers against flanking regions of alg5-A and as template, DNA isolated from background strain cells (lane 1), from plasmid-incorporating cells (lane 2), or from cells in which the plasmid and native alg5-A had been expelled (lane 3). (B) RT-PCR was performed using primers against alg5-A (upper panel) or alg5-B (lower panel). As template, DNA (lane 2), RNA (lane 3) or cDNA (lane 4) from cells lacking alg5-A was used. DNA from background cells served as template in control reactions (lane 1).

Lipid modification in Archaea

H. volcanii cells are surrounded by a protein shell solely comprised of the S-layer glycoprotein. Using pulse chase radiolabelling and cell fractionation studies, we have revealed that newly-synthesized S-layer glycoprotein undergoes a maturation step following translocation of the protein across the plasma membrane. (Eichler, 2001; Konrad and Eichler, 2001). The processing step is due to a mevalonic acid-based lipid modification of the protein. Studies aimed at describing the mechanism of this post-translational modification are on-going. 

Maturation of the H. volcanii S-layer glycoprotein. Cells were [³⁵S]-metabolically radiolabeled and then chased with an excess of unlabeled methionine. Aliquots were removed immediately prior to and then at intervals following onset of the unlabeled methionine chase. The samples were then examined by 7.5% SDS-PAGE and fluorography.

2D gel electrophoresis reveals differences in the hydrophobicity of the mature and immature versions of the S-layer glycoprotein. An aliquot of cells subjected to pulse chase radiolabelling was removed 4 min after the onset of chase and examined by non-denaturing gel electrophoresis in the presence of 0.5% Triton X-100. The gel lane was cut and applied horizontally to the top of the 3% stacking gel of a 7.5% separating SDS-PAGE gel and electrophoresis was performed as usual. The gel was then dried and examined by fluorography. The directions of electrophoresis under non-denaturing and denaturing (i.e., SDS-PAGE) are shown at the top and to the left of the gel, respectively. Arrows depict the positions of the mature and immature S-layer glycoprotein.

The H. volcanii S-layer glycoprotein can be labeled by [³H] mevalonic acid. H. volcanii cells were grown in the presence of [³H] mevalonic acid. Aliquots were then removed and processed for SDS-PAGE and examined by fluorography (lane 1), immunoblotting with anti-S-layer glycoprotein antibodies (lane 2) or PAS staining (lane 3). The arrow on the left depicts the position of the S-layer glycoprotein, while the position of molecular weight markers are shown on the right.

PROTEIN TRANSLOCATION IN ARCHAEA

In Archaea, a variety of extracytoplasmic proteins must be translocated into and across the plasma membrane. These include membrane proteins, secreted enzymes and the components of the protein based surface layer found in numerous archaeal species. However, in contrast to the well-studied phenomena of protein translocation across the bacterial plasma membrane or across the eukaryotic ER membrane (the topological homologue of the prokaryotic plasma membrane), very little is understood of how proteins cross the plasma membrane of Archaea. Our research is aimed at elucidating the various processes, components and mechanisms involved in protein translocation across the archaeal plasma membrane.

Dissection of the archaeal SRP pathway

The challenge of transfering proteins across phospholipid-based membranes begins with correct targetting to proteinaceous translocation complexes embedded in the membrane. Eukarya, Bacteria and Archaea all contain components of the signal recognition particle (SRP), a ribonucleoprotein complex involved in the delivery of nascent proteins to translocation sites. In Eukarya, the SRP pathway is relatively well-characterized, while significant strides in elucidating the role of bacterial SRP have been realized in recent years. In contrast, little is known about the SRP pathway in Archaea (Zwieb and Eichler, 2002). Accordingly, our group is looking at various aspects of archaeal SRP composition, assembly and function (Tozik et al., 2002).

Co-translational membrane protein in insertion in Archaea

In H. volcanii, ribosomes are found in the cytoplasm and membrane-bound at similar levels. Transformation of H. volcanii to express chimeras of the translocon components SecY and SecE fused to a cellulose-binding domain substantially decreased ribosomal membrane-binding, relative to non-transformed cells, likely due to steric hinderance by the cellulose-binding domain. Decreased ribosomal binding selectively affected membrane protein levels, suggesting that membrane insertion occurs co-translationally in Archaea (Ring and Eichler, 2004).

Nascent membrane protein biogenesis is reduced in CBD-SecE- and CBD-SecY-expressing H. volcanii cells. A. The radioactivity associated with the soluble and membrane protein fractions of metabolically [³⁵S]cysteine/methionine radiolabelled background (WT), CBD-SecE- and CBD-SecY-expressing H. volcanii cells, grown to identical densities, was assessed by ß-scintillation counting (A) or SDS-PAGE and fluorography (B). A. The radioactivity present in 5 µl samples was measured.