1 CHAPTER 1 INTRODUCTION 1.1 POST TRANSLATIONAL MODIFICATION OF PROTEINS The diversity in nature’s repertoire of proteins is contributed by the differences in arrangement of amino acids. This diversity is enhanced by post translational modifications to perform several biological functions. Post translational modifications are covalent processing events that alter the properties of a protein by proteolytic cleavage or by addition of modifying groups to one or more amino acids. Such processing events modulate biological processes by influencing protein activity, localization, turnover, and interactions with other proteins (Mann and Jensen 2003). The proteome diversification by covalent modification occurs in both prokaryotes and eukaryotes; in latter it is much more extensive in terms of types of modifications and frequency of occurrence (Walsh et al 2005). The most common types of covalent protein modifications include; phosphorylation, glycosylation, disulfide bond formation, acylation (such as ε-N-acetylation, N-myristoylation, S-palmitoylation, mono- and polyubiquitylation) and alkylation (such as N-methylation and S-prenylation) (Walsh et al 2005). Apart from the above well-characterized and abundant covalent modifications, there are many additional classes of enzymatic modification of proteins that expand the metabolic and signaling capacities of organisms. These include, protein hydroxylation, sulfur transfer, ADP-ribosylation, carboxylation, phosphopantetheinylation etc (Walsh et al 2005, Yarbrough and Orth 2009, Walsh and Jeffries 2006).
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1
CHAPTER 1
INTRODUCTION
1.1 POST TRANSLATIONAL MODIFICATION OF PROTEINS
The diversity in nature’s repertoire of proteins is contributed by the
differences in arrangement of amino acids. This diversity is enhanced by post
translational modifications to perform several biological functions. Post
translational modifications are covalent processing events that alter the
properties of a protein by proteolytic cleavage or by addition of modifying
groups to one or more amino acids. Such processing events modulate
biological processes by influencing protein activity, localization, turnover,
and interactions with other proteins (Mann and Jensen 2003). The proteome
diversification by covalent modification occurs in both prokaryotes and
eukaryotes; in latter it is much more extensive in terms of types of
modifications and frequency of occurrence (Walsh et al 2005). The most
common types of covalent protein modifications include; phosphorylation,
glycosylation, disulfide bond formation, acylation (such as ε-N-acetylation,
N-myristoylation, S-palmitoylation, mono- and polyubiquitylation) and
alkylation (such as N-methylation and S-prenylation) (Walsh et al 2005).
Apart from the above well-characterized and abundant covalent
modifications, there are many additional classes of enzymatic modification of
proteins that expand the metabolic and signaling capacities of organisms.
These include, protein hydroxylation, sulfur transfer, ADP-ribosylation,
carboxylation, phosphopantetheinylation etc (Walsh et al 2005, Yarbrough
and Orth 2009, Walsh and Jeffries 2006).
2
1.2 COMMON POST TRANSLATIONAL MODIFICATIONS –
GLYCOSYLATION, PHOSPHORYLATION AND LIPID
MODIFICATION OF PROTEINS
Of the several post-translational modifications, glycosylation is the
most common post-translational process in eukaryotes and accounts to 1-2%
of the proteins encoded by the human genome (Walsh and Jeffries, 2006).
Most of the cell surface and secreted proteins are glycoproteins (Ashford and
Platt 1999). In this type of modification, oligosaccharides are attached
co-translationally to specific asparagine (N-linked) or serine/threonine
(O-linked) residues; for N-linked glycosylation the consensus sequence
Asn-X-Ser/Thr is essential, (where X can be any amino acid except proline),
whereas sites of O-glycosylation show no specific amino acid sequence
(Ashford and Platt 1999). The sugar moieties of glycoproteins affect both the
structural and functional properties of the protein, such as protein folding and
conformation, stability to denaturation, solubility and resistance to proteolysis
as well as key biological properties such as receptor binding, modulation of
enzyme activity and cellular recognition events (Walsh and Jeffries 2006).
Another important post translational modification is
phosphoryalation of proteins, generally recognized as a fundamental
mechanism by which the intracellular events are modulated (Morandell et al
2006). The process is reversible, enabling the cells to respond to myriad
signals. In eukaryotes, phosphorylation usually occurs on Ser, Thr, and Tyr
residues whereas, in prokaryotes it occurs on the basic amino acid residues of
His or Arg or Lys. The reversible phosphorylation in many enzymes and
receptors results in a conformational change, causing them to become either
activated or deactivated, and thereby controlling protein activity within the
cells. For example, caspases, the key degradative enzymes that function in the
apoptotic process are activated upon phosphorylation.
3
Covalent attachment of lipids to proteins is an essential post
translational mechanism occurring in both eukaryotes and prokaryotes. It was
first demonstrated from the studies in Escherichia coli murein lipoprotein by
Braun and Rehn 1969. The discovery was soon followed by the identification
of fatty acids linked to viral glycoproteins, fungal mating factors and to
GTP-binding proteins (Baumann and Menon 1985). The eukaryotic lipid
modification of proteins attracted most of the attention and was intensely
studied (Yalovsky et al 1999). Eukaryotic lipidation ranges from addition of
myristyl, palmitoyl, diphatnyl or cholesterol moieties conferring wide range
of lipophilicity. These can be added at the amino terminus, the carboxy
terminus, or at internal residues via ester, thioester, thioether, or amide bonds;
or through mediating elements, activated intermediary carrier like acyl carrier
protein also take part in lipid acylation (Walsh et al 2005). The following is a
brief account of our current understanding of protein lipidation.
1.2.1 Eukaryotic Lipid Modification
Unlike prokaryotes, in eukaryotes the lipid modification is diverse
with 10-50% of all proteins been possibly modified by lipids belonging to
isoprenoids (15-carbon farnesyl or 20-carbon geranylgeranyl groups) or
saturated fatty acyl groups (palmitoyl, myristoyl) or
glycosylphosphatidylinositol (GPI) (Hooper and Jeffrey Mcilhinney 1999).
These lipids tether the soluble proteins to membranes and allow protein-
protein interactions and transduction of signals. Lipoproteins have also been
implicated in a variety of other cellular and extracellular events like
embryogenesis, pattern formation, protein trafficking through the secretory
pathway and evasion of the immune response by infectious parasites
(Yalovsky et al 1999). The different types of lipid modification of proteins
seen in eukaryotes are briefly described as under.
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1.2.1.1 Prenylation
Among all the lipid modification mechanisms in eukaryotes,
prenylation of proteins is extensively studied (Gelb et al 2006). Anchoring
proteins to cellular membrane aids in several protein-protein interactions that
mediate signals for growth from cell surface receptors to nuclear transcription
factors (Yalovsky et al 1999, Gelb et al 2006). In, protein prenylation either a
farnesyl or a geranyl-geranyl moiety is transferred to C-terminal cysteine of
the target protein. Three enzymes that carry out prenylation are protein
farnesyltransferase (PTase), protein geranylgeranyltransferase type I
(GGTase-I) and protein geranylgeranyltransferase type II (GGTase-II), also
known as Rab GGTase (Zhang and Casey 1996, Hougland and Fierke 2009).
Protein prenyltransferases recognize the “CaaX” box, at the
c-terminal, which is the signature and transfers a prenyl group from either
farnesyl pyrophosphate or geranylgeranyl phosphate to the sulfhydryl group
of cysteine (Zhang and Casey 1996). Subsequently, last three amino acids,
two aliphatic and the C-terminal residue are removed by a prenyl protein–
specific endoprotease and the α-carboxyl group of prenylated cysteine is
methylated by a prenyl protein–specific methyltransferase. Farnesyl
transferases recognizes CaaX boxes where X is Met, Ser, Gln, Ala, or Cys,
whereas geranylgeranyl transferase-I recognizes CaaX boxes with X as Leu or
Glu and transfers geranyl geranyl groups to the cysteine. GGTases II transfers
two geranylgeranyl groups, with each attached to separate cysteines, and in
these cases there is no C-terminal carboxyl methylation observed (Zhang and
Casey 1996).
1.2.1.2 Myristoylation
In myristoylation myristate, a relatively rare 14-carbon fatty acid is
transferred cotranslationally, from myristoyl-CoA to the amino group of
5
N-terminal glycine residue of the target protein by the enzyme myristoyl-
CoA: protein N-myristoyltransferase (NMT) (Resh 1994). Myristate can be
attached to the N-terminal glycine in synthetic peptides. Myristoylated
proteins play a vital role in membrane targeting and signal transduction (Resh
1994).
1.2.1.3 Cholesterol Modification
It is a C-terminal posttranslational modification of a family of
signaling proteins referred to as hedgehog (Hh) proteins found in insects,
vertebrates, and other multicellular organisms (Mann and Beachy 2000).
These are involved in the patterning of diverse tissues during development.
Addition of cholesterol to Hh proteins proceeds via an autoproteolytic internal
cleavage reaction at the -Gly-Cys-Phe- tripeptide motif, characteristic of Hh
precursors and attachment of cholesterol to the C-terminal Gly (Mann and
(Lgt) catalyzes the first committed step of bacterial lipoprotein biosynthetic
pathway. The enzyme transfers the diacylglyceryl moiety of
phosphatidylglycerol to thiol group of the invariant cysteine in the lipobox of
prolipoproteins with concomitant release of glycerol-1-phosphate (Sankaran
and Wu 1994). A temperature sensitive (ts) mutant of Salmonella
typhimurium accumulated unmodified prolipoprotein in the cytoplasm at 42ºC
but not at 30ºC. Sequencing of the complementing 1.4-kilobase DNA insert
from S. typhimurium revealed an ORF of 291 amino acids, which is
immediately 5’ to the thyA gene and allelic to umpA of E. coli (Gan et al
1993).
After identifying the role of Lgt in bacterial lipoprotein
biosynthesis by Sankaran and Wu (1994) much of the research was carried
out to understand its structure-function relationship. Analysis of the primary
sequences of Lgt from phylogenetically distant species, such as Escherichia
coli, Salmonella typhimurium, Staphylococcus aureus and Haemophilus
influenzae revealed a significant degree of homology and conservation with
about 24% identity and 47% similarity (Gan et al 1995). The alignment of Lgt
sequences from phylogenetically distant species, such as Escherichia coli,
Salmonella typhimurium, Staphylococcus aureus and Haemophilus influenzae
revealed a conserved region of 103-HGGLIG-108, indicating its possible
involvement in active site (Qi et al 1995). The enzyme contained hydrophobic
segments interspersed with charged hydrophilic segments rich in Arg, among
Gram-negative organisms, Arg and Lys in Gram-positives, thus was deduced
with a pI value of 10.4. The enzyme was found to be inactivated with
diethylpyrocarbonate with a second-order rate constant of 18.6 M-1 s-1, and
this inactivation was reversible with hydroxylamine at pH 7, thus pointing
18
towards the involvement of a single modifiable residue, His or Tyr in its
activity. Accordingly, site-directed mutagenesis studies indicated role of
His-103 and Tyr-235 was crucial for Lgt activity. Consequently, deletion or
modification of these residues inactivated the enzyme (Sankaran et al 1997).
Role of lgt in growth and viability of bacteria was understood from
mutational studies carried out in lgt. The lgt null mutants in Gram-negatives
like E. coli and Salmonella were lethal (Qi et al 1995) unlike Gram-positives
that remained viable (Leskela et al 1999). This indispensability of Lgt in
Gram-negative bacteria has proscribed the study of virulence of lipoprotein-
processing mutants of Gram-negative pathogens. However, with several
Gram-positive bacteria as pathogens, implications from lgt mutants of such
pathogens revealed that not all cases showed attenuation of virulence (Leskela
et al 1999, Pettit et al 2001, Stoll et al 2005). Deletion of lgt in Listeria
monocytogenes caused impaired intracellular growth in human epithelial
(Caco-2) and mouse fibroblast (3T3) cell lines (Baumgärtner et al 2007).
Similarly, lgt mutants of S. agalactiae (Bray et al 2009, Henneke et al 2006)
and Staphylococcus aureus (Wardenburg et al 2006) showed hypervirulent
phenotypes in mouse models of infection. Thus, in lgt mutants there might be
a strain-specific balance between effects on immune activation and the
functional compromisation because of the loss of lipoprotein lipidation.
In a global topology analysis of the Escherichia coli inner
membrane proteome, Daley et al showed that Lgt is a transmembrane protein
(Daley et al 2005). However, based on a simple, precise radioactive assay, Lgt
was found to be associated to the inner-membrane peripherally (Selvan and
Sankaran 2008).
19
1.3.3.2 Lipoprotein signal peptidase
Among the enzymes involved in lipoprotein biosynthesis,
lipoprotein signal peptidase (Lsp) is the first enzyme to be identified and
studied in greater detail (Dev and Ray 1984). Lsp, a specific endopeptidase
recognizes diacylglyceryl modified prolipoprotein and cleaves the signal
peptide resulting in apolipoprotein (Sankaran and Wu 1995). The
identification of a fungal penta-peptide antibiotic, globomycin and its ability
to inhibit the processing of murein prolipoprotein to a lipoprotein is
considered as one of the significant contributions towards the understanding
of lipoprotein biosynthetic pathway in bacteria (Inukai et al 1978).
Globomycin-treated cells arrested translocation of Lpp to outer membrane
and its lipid-modified precursor accumulated in the inner membrane to the
accumulated precursors contained covalently linked glyceride (Hussain et al
1980).
The involvement of an exclusive signal peptidase for the cleavage
of lipoprotein signal sequence was identified in 1982 by Tokunaga et al.
Around the same time, the requirement of diacylglyceryl modified
prolipoprotein as a prerequisite for lipoprotein-specific signal peptidase was
demonstrated. With the knowledge that over-expression of lipoprotein signal
peptidase results in increased globomycin resistance, a clone containing
plasmid pLC3-13 was isolated and subcloned into pBR322 to generate
plasmid pMT52 (Tokunaga et al 1983, Yamagata et al 1983). This plasmid
was used to complement the temperature sensitive mutant of lipoprotein
signal peptidase in E. coli. This enabled mapping of the lsp gene between
0.5 to 0.6 min of E. coli genome (Regue et al 1984, Tokunaga et al 1985). The
amino acid sequence of the Lsp, as deduced contained 164 amino acids with a
molecular weight of 18 kDa. Lsp was deduced as an integral membrane
protein with four membrane-spanning segments connected by two periplasmic
20
loops and one positively charged cytoplasmic loop (Munoa et al 1991). Lsp
was also reported to be a novel aspartic protease (Sankaran and Wu 1995).
A biochemical assay for Lsp was developed by Dev and Ray in
1984. The commonly used [35S]-labeled diacylglyceryl modified
prolipoprotein was used as the substrate prepared from globomycin-treated
E. coli B cells. The assay also demonstrated that globomycin inhibited the
prolipoprotein signal peptidase in a non-competitive manner with a Ki value
of 36nM (Dev et al 1985). It was recently reported that Lsp can cleave even
unmodified prolipoprotein substrates in Listeria monocytogenes, indicating
perhaps the pathway does not follow a sequence always (Baumgärtner et al
2007). Likewise, a Streptococcus agalactiae lgt mutant revealed cleavage of
the ScaA lipoprotein precursor at the Lsp cleavage site in indicating its
activity towards unmodified forms in some Gram-positive bacteria (Bray et al
2009). Lsp mutants of several Gram-positive pathogens have shown
attenuation of virulence (Zhao and Wu 1992, Mei et al 1997, Tjalsma et al
1999); Lsp mutants of Mycobacterium tuberculosis showed reduced growth in
macrophages when cultured in vitro (Sander et al 2004). Failure of Lsp
mutant to activate immune responses via TLR2 was identified with lsp
mutants of Streptococcus agalactiae, Streptococcus equi and Streptococcus
pneumonia (Henneke et al 2006).
1.3.3.3 Phospholipid:apolipoprotein transacylase
Phospholipid:apolipoprotein transacylase (Lnt) catalyzes the
transfer of an acyl moiety to the amino group of the apolipoprotein through
amide linkage and concomitant release of lysophospholipid. The acyl donor
for this reaction could be any phospholipid present in the inner membrane
(Sankaran et al 2005).
21
This enzyme catalyzing the conversion of apolipoprotein to mature
lipoprotein, was detected by an in vitro assay using [35S]methionine-labeled
apolipoprotein as the substrate. The mature lipoprotein generated following
enzymatic conversion of apolipoprotein was estimated by densitometric
scanning of the autoradiogram (Gupta and Wu 1991). Further, studies
revealed phosphatidylethanolamine is not essential for the N-acylation of
apolipoprotein and subsequent formation of lipoprotein. But, other major
phospholipids such as phosphatidylglycerol and cardiolipin could also serve
as the donor of fatty acid in N-acylation of apolipoproteins (Gupta et al 1991).
Gupta et al isolated a temperature sensitive mutant of Salmonella
typhimurium, SE5312, which accumulated apolipoprotein at 42°C. The
mutant defective in N-acyl transferase activity was complemented by a gene
allelic to cutE of E. coli (Gupta et al 1995). Mapping of this mutation placed
the lnt gene in 14-17 min of Salmonella typhimurium chromosome (Rogers
et al 1991). The lethality due to loss of Lnt activity was reported to be due to
the retention of apo-Lpp in the cytoplasmic membrane, implicating Lnt
activity is essential for proper localization of outer membrane lipoproteins.
Although biochemical analysis of Braun’s lipoprotein expressed in
Bacillus subtilis and lipoprotein preparations from Staphylococcus aureus
revealed N-acylation, BLAST search for homologues of Lnt could not be
identified in Gram-positives like Firmicutes (Hayashi et al 1985, Navarre et al
1996) However, Streptomyces coelicolor revealed homologues of Lnt but the
gene (SCO1336) failed to complement the activity in an E. coli lnt depletion
strain. Topology mapping of Lnt with -galactosidase and alkaline
phosphatase fusions indicated the presence of six membrane-spanning
segments (Robichon et al 2004). The deduced amino acid sequence revealed
512 amino acids and an estimated molecular mass of 56 kDa. The optimum
pH was found to be in the range of 6.5 to 7.4 and an appreciable activity was
22
reported upto 60oC (Sankaran et al 1995). Lnt, classified as a member of the
nitrilase superfamily, contains a common Glu-Lys-Cys catalytic triad (Pace
and Brenner 2001). Seven conserved residues for Lnt were identified based on
which a structural model was also predicted. The essential residues were, the
potential catalytic triad formed by E267-K335-C387, Y388 and E389
comprising the hydrophobic pocket, which also has the active site and W237 -
E343, which are away from the active site, are expected to open and close
upon the binding and release of phospholipid and/or apolipoprotein (Vidal-
Ingigliardi et al 2007).
1.3.4 Translocation of Bacterial Lipoproteins Across Inner
Membrane
Secretory proteins are synthesized in the cytoplasm to reach their
destination outside the cytoplasm, these proteins need to be recognized and
targeted by the protein secretion system. The major route for protein transport
across cytoplasm is through ‘Sec’ machinery translocation, in which secretory
proteins are translocated in an unfolded state (Pugsley 1993). Another
recently identified protein translocation system, Twin Arginine Translocase
(TAT) Pathway exclusively exports pre-folded or fast-folding secretory
proteins (Berks 1996, Sargent et al 1998, Thomas et al 2001).
Bacterial prolipoproteins, which are synthesized within the
cytoplasm, are all known to be translocated via Sec (Sugai and Wu 1992).
As, the enzymes for modification and processing are present in the inner
membrane, the association between Sec and lipoprotein biosynthetic
machineries had been of interest, but not adequately probed. However, it has
been shown that mutants impaired in secretion were also found impaired in
lipid modification (Sugai and Wu 1992). Although, TAT is implicated for
translocation of prolipoproteins, it has not been adequately studied and
23
understood (Lee et al 2006). A detailed account of both Sec and TAT
pathways and its role in translocation of bacterial prolipoproteins are given
below.
1.3.4.1 The common Sec pathway
The Sec pathway is the only known conserved protein translocation
pathway in all the three domains of life. The pathway involves a series of
steps to export proteins in an unfolded manner across the cytoplasm, which is either post-translational or co-translational (Mitra et al 2006). In bacteria, the
Sec translocase is a stable heterotrimeric organization, SecYEG, which
comprises three integral membrane proteins SecY, SecE and SecG. This complex associates with the auxiliary protein complex, SecDFYajC and
YidC. SecA, a dimeric ATPase is located at the cytoplasmic side of SecYEG
complex (Mitra et al 2006). SecB is an acidic homo tetrameric chaperone protein organized as dimer of dimers and wraps around pre-protein and
prevents premature folding of the protein (Driessen 2001). The nascent
polypeptide chain emerging from the ribosome is mostly routed to the Sec Translocase in SecB-dependent manner. In SecB-independent targeting, the
pre-proteins are translocated as ribosome-bound nascent chains (RNCs) by
the signal recognition particle (SRP) (Mitra et al 2006).
SecB-bound pre-protein, facilitates electrostatic interaction between SecB and SecYEG-associated SecA. The interaction allows transfer of
pre-protein from SecB to SecA upon ATP binding the interface of the two
nucleotide binding folds (NBF1 and NBF2) of SecA (Fekkes et al 1998). The energy from ATP hydrolysis together with proton motive force facilitates
translocation of pre-proteins through SecYEG core (Mitra et al 2006). In
co-translational translocation, SRP interacts with pre-proteins to form ribosome nascent chain complex (RNC) .The complex is targeted to
SRP-receptor; FtsY, which in turn is bound to translocation-competent
SecYEG. Upon interaction with the receptor, RNC is transferred to SecYEG
24
core, which requires GTP hydrolysis (Driessen 2001). The precursor proteins
reaching this core are speculated to be pumped across the membrane barrier by utilizing proton motive force (Driessen 2001, Mitra et al 2006) (Figure 1.6).
Figure 1.6 Schematic overview of Sec translocation (Keyzer et al 2003)
showing the association of nascent polypeptide of secretory
protein with ‘Sec’ complex
1.3.4.2 Discovery of Twin Arginine Translocase (TAT) pathway
The Twin Arginine Translocase (TAT) pathway was first
discovered only recently (1995) in plant thylakoids (Chaddock et al 1995,
Berks 1996, Clark and Theg 1998). It functions in a radically different way to
that of the Sec translocase. The translocation is independent of nucleotide
triphosphate hydrolysis and depends solely on proton gradient hence, referred
as ∆pH pathway (Cline et al 1992, Alder and Theg 2003). The extensive
studies on the new mechanism of export in thylakoids revealed that the signal
peptides of the target proteins exported contained a common and essential
twin-arginine motif preceding the hydrophobic region (Chaddock et al 1995).
Berks (1996) observed that certain bacterial periplasmic proteins contained a
25
conserved twin arginine motif at the n-h boundary as in thylakoid
pre-proteins, implicating the existence of ∆pH-driven translocation in
bacteria. One such cofactor requiring enzyme, Trimethylamine N-oxide
reductase (TMAO reductase) was found to fold only in the presence of
molybdenum and then exported to periplasm in a Sec-independent manner
(Santini et al 1998). Around the same time, the first component of
∆pH-system was identified in maize and later its homologs were identified in
E. coli and were found to encode two distinct genes, one of them belonged to
a four-gene operon, and the other was unlinked (Sargent et al 1998). The
products of these genes were found to be required for the Sec-independent
export of a range of proteins with twin-arginine motif in its signal sequences
(Bogsch et al 1997, Sargent et al 1998). The genes were named as tatA in
putative tatABCDE operon and tatE (Sargent et al 1998, Hicks et al 2003).
Later, Hynds and coworkers (1998) reported the ability of ∆pH-pathway to
export tightly folded proteins in thylakoids.
Characteristic signal sequences of proteins translocated via the TAT
pathway: Signal sequences that target proteins to the TAT machinery
conform to overall tripartite structure but have additional distinct features that
delineates from Sec-signal peptides. The striking feature is the presence of
consensus motif –S/T-R-R- X- F -L- at the n-h boundary with invariant
consecutive Arg residues are almost invariant, X is any polar amino acid
(Berks 1996) (Figure 1.7). Substitution of either arginine residues with lysine
appears to block transport. Nevertheless, in rare cases it has been observes
putative TAT substrates. Ser, Thr, Gly, Asp and Asn occupy -1 position
predominately, with serine occurring in more than 50% of the known
sequences (Lee et al 2006). Site-directed mutagenesis of conserved residues
in the motif revealed Phe and to a lesser extent Leu is important for TAT
targeting (Stanley et al 2000). The TAT signal sequences are longer with
28-56 amino acids compared to18-26 amino acids of Sec signal sequences
26
(Berks 1996). The additional length in TAT signal sequences is largely due to
extended n-region (Berks 1996). The h-region is less hydrophobic than that of
Sec signal peptides due to a higher occurrence of Gly and Thr and a
significantly lower abundance of Leu residues (Berks 1996, Cristobal et al
1999). The c-region is characterized by the presence of basic amino acids
whereas such a feature is uncommon among Sec signal sequences. Actually
this feature along with degree of hydrophobicity of h-region acts as
“sec-avoidance” signal (Berks 1996, Bogsch et al 1997).
Figure 1.7 Features of a typical TAT signal peptide from
E. coli, TorA highlighting the characteristic TAT-
recognition sequence between n and h regions, and the
cleavage region preceded with positively charged residues,
the ‘Sec’-avoidance signal (Lee et al 2006)
Components of TAT Pathway: In E. coli, four genes tatA, tatB, tatC and
tatE were identified to encode integral membrane proteins constituting the
TAT components (Lee et al 2006). The tatA, tatB and tatC genes form an
operon with a fourth promoter-distal gene, tatD, whereas tatE is
monocistronic (TatD, a soluble protein with DNase activity was later found to
have no role in TAT pathway) (Sargent et al 1998, Wexler et al 2000). tatE is
a cryptic gene duplication of tatA and codes for the same functional protein.
In Gram-positives, Gram-negatives, tatB gene is missing; it has only
homologues of tatA and tatC genes (Berks et al 2003).
27
In E. coli the minimum TAT components required for TAT
translocation are TatA, TatB and TatC (Tha4, Hcf106, and cpTatC in
chloroplasts, respectively) (Behrendt et al 2004, Lee et al 2006). TatA and
TatB respectively are 9.6kDa and 18.4kDa proteins with a hydrophobic
transmembrane α-helix at their N-terminus followed by an amphipathic
α-helix localized at the cytoplasmic side of the membrane. TatC is a 28.9-kDa
protein with six TM regions (Allen et al 2002). It is an essential component of
TAT system, as deletion mutants of tatC completely abolished
TAT-dependent transport (Bogsch et al 1998, Allen et al 2002). Detergent-
solubilized membranes of E. coli cells over expressing TAT components
revealed complexes of ~600 kDa to contain varying numbers of TatA (4 to
100; average 25) but with , a strict stoichiometric ratio of 1:1 of TatB and
TatC (de Leeuw et al 2002, Oates et al 2003).
Alami and coworkers (2003) used the site-specific cross-linking
studies to reveal the interaction of TatC interacts with the consensus -RR-
motif and the interaction of TatB with the entire length of the signal sequence
along the hydrophobic region extending to adjacent mature region. The
studies thus revealed that TatC formed the primary recognition site of TAT
Translocase. It was demonstrated that TatA transiently associated with TatBC
only in presence of a TAT-substrate and transmembrane proton gradient
(Alami et al 2003). TatA polymerizes on binding to TatBC complex at the
time of translocation to form a translocation channel of variable pore size and
can accommodate folded substrates upto 70 Å. To prevent ion leakage the
TatA protomers form a tight seal around the substrate and exports the
substrate across the membrane in an iris-type fashion (Gohlke et al 2005)
(Figure 1.8).
28
Figure 1.8 Schematic overview of TAT pathway showing Tat
components and translocation of pre-folded secretory
proteins (Lee et al 2006)
Substrates of TAT pathway: The TAT pathway transports substrates that
require folding in cytoplasm. Majority of the TAT-dependent proteins were
identified as co-factor requiring proteins such as, hydrogenases, formate