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Signal Peptides 307
14.4.2.2 Specificity of Type I Signal Peptidases
As noted above, signal peptides have a weak consensus pattern around their cleavage
site known as the (–3, –1) rule. 153 Namely, the residues at positions –3 and –1 from
the cleavage site are small (and neutral) such as alanine in most cases. In eukaryotes,
this requirement is less stringent, allowing glycines and serines as well as alanines;
it is more stringent in thylakoid membrane. 169,172,173 With experimental analyses, this
rule was confirmed as the substrate specificity of type I signal peptidases. However,
because the consensus pattern is so weak, many other potential consensus sites could
be around the authentic site in a preprotein. More importantly, it seems difficult to
discriminate signal-anchored proteins from proteins with cleavable signal peptides
by mere existence of such a consensus pattern. 151
Several experiments show that the degree of hydrophobicity in h-region affects
the cleavage. 174 For example, signal peptides become uncleavable when their hregion
is artificially extended. 175 The three-dimensional structure of a catalytic Nterminal
fragment of E. coli type I signal peptidase gives some hints on this
enigma. 176,177 The structure shows that the c-region must be in an extended conformation;
since transmembrane segments in integral membrane proteins are generally
longer and more hydrophobic than the h-region of signal peptides, these segments
may not be able to be properly positioned at the catalytic site. However, this idea
was challenged by a recent experiment in which both the lack of transmembrane
region of signal peptidases and mutations in the h-region of signal peptides did not
affect the correct cleavage site selection. 178
Structures of the mature protein can also affect the cleavage. For example,
removal of N-terminal cytoplasmic tail of a type II membrane anchor protein could
lead to the cleavage of its transmembrane region; 179 in another example, the transmembrane
region near the C terminus can affect the cleavage of signal peptides. 180
Thus, the prediction of signal-anchored proteins is still a difficult problem (see
Section 14.5.1).
14.4.2.3 Other Types of Signal Peptidases
Type II signal peptidases (signal peptidases II) are found in eubacteria but not in
archaea. These peptidases cleave the signal peptides of so-called lipoproteins (lipidmodified
proteins) after the lipid modification. They show a clearer substrate specificity
than type I peptidases: the lipobox, is typically represented as LA(G/A) | C,
where | denotes the cleavage site. 181,182 The cysteine residue is linked by a fatty acid
and the protein is anchored at the membrane with this lipid molecule. The leucine at
the –3 position may be other large hydrophobic residues. In B. subtilis, signal peptidase
II is required for efficient secretion of α-amylase, a nonlipoprotein. 183 Therefore, the
substrate specificity of signal peptidase II may not be restricted to lipoproteins.
The third type of signal peptidase is PilD (XcpA), which specifically cleaves
preproteins related to pilus biogenesis, 184-186 which is related to the so-called type II
protein export pathway. 187 In this pathway, the leader sequences are exceptional in
that they are basic and short. PilD not only cleaves these leader peptides but also
subsequently monomethylates the newly exposed N-terminal phenylalanine.