Biochimie 114 (2015) 134e146

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Biochimie
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Review

N-terminal protein modifications: Bringing back into play the

ribosome
Carmela Giglione a, b, *, Sonia Fieulaine a, b, Thierry Meinnel a, b, *
a
CNRS, Institut des Sciences du V
eg
^t 23A, F-91198 Gif sur Yvette, France
etal, 1 Avenue de la Terrasse, Ba
b
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Universit

e Paris-Sud, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: N-terminal protein modifications correspond to the first modifications which in principle any protein
Received 23 September 2014 may undergo, before translation is completed by the ribosome. This class of essential modifications can
Accepted 10 November 2014 have different nature or function and be catalyzed by a variety of dedicated enzymes. Here, we review
Available online 18 November 2014
the current state of the major N-terminal co-translational modifications, with a particular emphasis to
their catalysts, which belong to metalloprotease and acyltransferase clans. The earliest of these modi-
Keywords:
fications corresponds to the N-terminal methionine excision, an ubiquitous and essential process leading
Co-translational modifications
to the removal of the first methionine. N-alpha acetylation occurs also in all Kingdoms although its
N-terminal
Peptide deformylase
extent appears to be significantly increased in higher eukaryotes. Finally, N-myristoylation is a crucial
Methionine aminopeptidase pathway existing only in eukaryotes. Recent studies dealing on how some of these co-translational
N-a-acetyltransferase modifiers might work in close vicinity of the ribosome is starting to provide new information on when
N-myristoyltransferase these modifications exactly take place on the elongating nascent chain and the interplay with other
ribosome biogenesis factors taking in charge the nascent chains. Here a comprehensive overview of the
recent advances in the field of N-terminal protein modifications is given.
© 2014 Elsevier B.V. and Socie
te

Française de Biochimie et Biologie Mole

culaire (SFBBM). All rights
reserved.

1. Introduction
It is recognized that the majority of proteins, to become func-
tional actives, needs to undergo several modifications which are
usually catalyzed by specific enzymes. More than 400 different
protein modifications have been identified so far [1]. These modi-
fications can be reversible or irreversible, thus escorting the protein
along its whole life cycle. Modifications affecting the protein N-
termini, collectively known as N-terminal protein modifications
(NPMs), are the earliest modifications which a protein undergoes.
They involve the N-terminal methionine excision process (NME)
and acylations corresponding to N-a-acetylation (NTA) and N-
myristoylation (MYR) (Fig. 1). These modifications can often be
followed by further reversible modifications such as phosphoryla-
tion or further acylations such as S-palmitoylation (PAL). These
second-site modifications participate in the regulatory mecha-
nisms associated to the NPM pathways. All NPMs are essential for
* Corresponding authors. 1 Avenue de la Terrasse, B^
at 23A, F-91198 Gif sur Yvette,
France.
E-mail addresses: Carmela.Giglione@isv.cnrs-gif.fr (C. Giglione), Thierry.
Meinnel@isv.cnrs-gif.fr (T. Meinnel).
normal growth and function in most organisms and affect a vari-
able number of proteins.
NPMs usually take place co-translationally when the protein is
still very short and yet bound to the ribosome. They are catalyzed by
ubiquitous and essential enzymes being part of the so-called ribo-
some-associated protein biogenesis factors (RPBs), which are non-
ribosomal proteins acting as soon as a nascent polypeptide reaches
the extremity of the ribosomal exit tunnel [2,3]. RPBs ensure a
number of co-translational events, including not only nascent pep-
tide chain modifications but also protein folding and/or trans-
location into various cellular compartments. Indeed, the ribosome
functions as a central hub for a group of a dozen RPBs which act
simultaneously or sequentially and ultimately define the functional
biomolecules that populate the cell. The highly regulated mecha-
nisms that govern the action of RPBs in space and time are still poorly
understood. Some models have recently appeared in the literature,
highlighting coordination or competition events between RPBs.
The interest of the scientific community for NPMs has been
strongly revived during the past decade, probably because i) ge-
nomics has provided further insights into the diversity and origin of
the enzymes involved in these modifications, ii) proteomics issues
are now often turning to protein modifications and iii) natural or

http://dx.doi.org/10.1016/j.biochi.2014.11.008
0300-9084/© 2014 Elsevier B.V. and Socie
te

Française de Biochimie et Biologie Mole

culaire (SFBBM). All rights reserved.
 C. Giglione et al. / Biochimie 114 (2015) 134e146 135

Fig. 1. The co-translational N-terminal protein modifications (NPMs). NPMs are catalyzed by several enzymes that act successively: peptide deformylase (PDF) and methionine
aminopeptidase (MetAP) which ensure the N-terminal methionine excision process (NME), followed by N-a-acetyltransferase (Nat) or N-myristoyltransferase (NMT) that acylate
proteins. Depending of the organism and cellular compartment, the sequence of NPMs is quite different, as well as the amount of modified proteins. The percentage displayed here
into brackets reflects the relative number of occurrences in a given proteome, knowing that in terms of protein accumulation the value can be significantly increased [see dis-
cussions in Ref. [67,80,173]]. For instance, NTA is known to reach 90% of the total protein mass in human cell extracts, while the value is lower in terms of diversity.

synthetic compounds targeting NPMs with antibiotic, antiparasitic,
antifungal or anti-cancer effects have been discovered. Despite that
the physiological function(s) of these NPMs remained for long time
largely unknown, remarkable efforts have been recently deployed
allowing a depth characterization of the co-translational modifi-
cations and important breakthroughs concerning the enzymes
involved and the role of these NPMs in protein fate have been
highlighted. The ways some of the enzymes involved in the process
of NPMs work and act in concert with other RPBs at the level of the
ribosome have been refined in recent times.
In this review, we provide an overview of the early N-terminal
protein modifications and we summarize and discuss the recent
data underlying the mechanistic details on how several of NPM
enzymes emerge to be involved in association with the ribosome.
and in the organelles [6e9]. These rules are conserved even when a
non-conventional initiation codon is used (i.e., different from the
classic AUG start codon, including GUG, AUU, CUG…). However, the
first Met is often specifically removed from proteins accumulating
at the steady state. Thus, the irreversible NME process corresponds
in bacteria and in the organelles of eukaryotes to the early removal
of the N-formyl group immediately followed by the removal (in
both bacteria and in eukaryotes) of the first Met. NME process is
ensured by two metalloenzymes known as peptide deformylases
(PDFs) and methionine aminopeptidases (MetAPs) which sequen-
tially remove the formyl group, when it is present, and the N-ter-
minal Met, respectively (Fig. 1).
2.1. Extent of the NME process

2. The NME process
Among NPMs, NME is the first occurring widespread protein
modification. To understand what the NME process is and what it
induces on protein N-termini, it should be remembered that the
initiation process of ribosome-dependent protein synthesis
strongly differs depending on the type of organisms or cellular
compartments thereof (i.e., cytoplasm, mitochondrion or plastid)
[4,5]. In archaea and the cytoplasm of eukaryotes, protein synthesis
always starts with an unblocked, so-called “free” methionine (Met),
whereas it starts with an N-formylated Met (Fo-Met) in Eubacteria
Between 30 and 60% of proteins are liberated of the N-terminal
Met, depending on the organism and cellular compartment [10]
(Fig. 1). More than 90% of characterized bacterial or chloroplast
encoded proteins undergo deformylation [This laboratory, unpub-
lished results and Ref. [11]], whereas the percentage of
mitochondria-encoded proteins undergoing deformylation is still
not clearly defined. Indeed, the number of mitochondrially-
encoded proteins is variable depending on the organism [e.g., 13
in human [12] and 33 in the model plant Arabidopsis thaliana [13]]
and because their high hydrophobicity the chemical characteriza-
tion has remained elusive for long time. By measuring the precise
136 C. Giglione et al. / Biochimie 114 (2015) 134e146

intact molecular masses of most of the 13 mitochondrial encoded
proteins from bovine heart it has been suggested that most likely
only the Cox III protein undergoes a complete NME process [12].
Nonetheless, it has been shown that the human mitochondrially
PDF is able to efficiently deformylate, at least in vitro 7, formylated
peptides derived from the N-terminus of mitochondrial encoded
proteins [14]. The sequencing of 8 of 32 mitochondrially encoded
plant proteins clearly reveals that all the sequenced proteins with
one exception having their N-Formyl-Met being removed [15].
2.2. Control and physiological roles of NME
NME is conserved from bacteria to higher eukaryotes and is
essential for normal growth and function in any organism investi-
gated [11]. Despite NME is still considered as a non-dynamic
imprinting modification of any proteome, many data highlighted
that the enzymes ensuring the process are tightly regulated during
development, tumorigenesis and in response to abiotic and biotic
stresses [16e23]. Nonetheless, a clear impact of these regulated
processes on the modification state of the entire proteome is not
directly evidenced so far. Why up to two-third of proteins of any
proteome undergo a very early NME process has remained elusive
for long time. More than 25 years ago NME was suggested to be
critical for protein half-life [24]. This is in line with the discovery
that NME modulates the lifespan of a reduced number of chloro-
plast key proteins, suggesting that NME might directly contribute to
protein stability, and that either removal or maintenance of the first
Met can be perceived as stabilizing or destabilizing signals,
respectively [25]. The recent extraordinary accomplishments in this
field provided strong support for the “protein stability” hypothesis,
bridging the gap between NME and protein half-life. Particularly, in
the context of the N-end rule, the best-characterized specific signal
for protein degradation [for reviews see Refs. [26,27]], the ambig-
uous role of the first Met as a destabilizing or stabilizing residue has
been recently elucidated and showed that the N-termini of proteins
starting with an N-alpha-acetylated Met (see next sections) or a
free Met may serve as the site of N-ubiquitination by regulation of
their steric shielding [25,28e30]. Through its global control of
protein half-life, NME was shown to fine tune the global gluta-
thione redox homeostasis at least in plants, yeast and Archaea [31].
2.3. Peptide deformylases
Peptide deformylase (PDF) is the first enzyme to modify the N-
terminal extremity of newly synthesized proteins. It is essential for
eubacteria [32,33] and leads to serious dysfunction if altered in
organelles [25,34e36]. PDFs are mostly monomeric hydrolases
belonging to the thermolysin-metzincin HEXXH motif containing
protein family, shown to absolutely require a metal cation bound to
either of the His residues of the motif for hydrolysis to occur [37,38].
Although believed to exist only in bacteria, during the last decade
PDFs were found in almost all organisms, including not only pro-
karyotes and most eukaryotes but also in viruses [39e42]. The low
sequence similarity observed for all identified PDFs contrasts with
the high evolutionary conservation of 3 short stretches of amino
acids (motifs I, II and III) building the whole active site of these
enzymes (Fig. 2A). A Cys residue crucial as third ligand of the metal
cation features in motif II. Motif I creates series of hydrogen bonds
made with a Gln side chain and peptide bond residues for the
formyl-Met substrates to properly and specifically align the active
site, where the Glu of Motif III and the metal cation are crucial for
hydrolysis to occur. Moreover, the 3D structures of many currently
available PDFs, coming from almost 30 different organisms, reveal
that they all share identical structure, the active site fold is similar
and that the main structural difference concerns the C-terminal
part. Thus, based upon structure and sequence similarity, PDFs are
divided into three groups (1, 2 and 3), with Type 1 including a
subgroup 1A and a subgroup 1B. Type 1B PDF is considered as the
structural PDF model, with a globular core and a C-terminal a-helix
[43,44] (Fig. 2B). PDFs of Type 2 possess several insertions within
the globular core, and harbor a totally different C-terminal ex-
tremity compared to Type 1B PDF [45] (Fig. 2B). Different to PDFs of
Type 1B that are found in bacteria and organelles, PDFs of Type 1A
are exclusively located in organelles [40,46e48]. They possess a
long insertion that partially clogs the active site entrance,
contributing to a narrow ligand binding site, and harbors a C-

Fig. 2. Sequence and structure of peptide deformylases (PDFs). PDFs are classified into three groups (types 1, 2 and 3) and two type 1 subgroups (1A and 1B). (A) Sequence
alignment around the three conserved motifs, with two representative sequences of each PDF type. Escherichia coli and Pseudomonas aeruginosa for type 1B; cyanophage S-SSM7
and viral 1502.1906 [39,41] for C-terminal truncated type 1B; Bacillus stearothermophilus and Staphylococcus aureus for type 2; Homo sapiens and Arabidopsis thaliana for type 1A;
Trypanosoma brucei and Leishmania major for type 3. The 38 and 90 last residues of Type 3 PDFs has been truncated (noted X38 and X90). Identical residues are boxed in red when
strictly conserved or red-colored when conserved in all sequences except for one or two sequences [174]. (B) Comparison of archetype PDF structures for types 1B, 2 and 1A.
Structures are directed towards the substrate binding cleft of the catalytic site, with the three characteristic motifs of PDF enzymes highlighted in magenta. The variable C-terminal
extremity is shown in green and specific insertions are highlighted in red or blue. The comparison of C-terminal extremity folding is shown through superimposition of the three
types of PDF structures with the globular core of Escherichia coli PDF represented as molecular surface (right).
 C. Giglione et al. / Biochimie 114 (2015) 134e146
Fig. 3. Domain architecture and structure of methionine aminopeptidases (MetAPs).
MetAPs are classified into two groups (types 1 and 2) and several subgroups (1a-d and
2a-b), distinguished by specific additional domains. (A) All MetAPs possess a common
catalytic domain (pink), in which MetAPs of type 2 harbors two insertions (designed as
N- and C-terminal insertions, shown in green and blue respectively). MetAP1a' from all
Streptococci and several Lactobacilli differs from the classical MetAP1a enzymes
through the presence of two insertions within the conserved catalytic domain.
Eukaryotic MetAPs display N-terminal extensions of variable length and sequences
(red). Ebp1 is a human protein related to type 2 MetAP group, i.e., with insertions in the
MetAP related catalytic domain. It also possesses a C-terminal domain, composed of
basic and acidic stretches. (B) Comparison of archetype MetAP structures for types 1a,
1b, 1c, 2a and 2b (E. coli, Homo sapiens, Mycobacterium tuberculosis, Pyrococcus
137
terminal extremity different to that found in Type 1B and 2 [14,47]
(Fig. 2B). This C-terminal part is totally absent or shorter in the
recent discovered viral PDFs, all belonging to the Type 1B subgroup
(Fig. 2A) [39,41,42,49]. Despite these differences at the C-terminus,
for one of these viral PDFs it has been recently shown to display a
classical PDF activity in vitro [41]. Type 3 PDFs, for which no 3D
structure is available to date, are also characterized by a much
longer C-terminal extension [50] (Fig. 2A), which is likely suggested
to fold as an a-helical structure (data not shown). Type 3 PDFs are
suggested to be inactive because they display substitutions in
crucial amino acids of motif I (Fig. 2A). To date, the exact role of
these inactive PDFs is not known, but it has been shown that
frequently the organisms exhibiting these inactive isoforms have
concomitantly active PDFs paralogs [51,52].
PDFs deformylate newly synthesized proteins in a co-
translational way. It has been shown that E. coli subtype 1B PDFs
interact with the ribosome in the exit tunnel region, with domain I
of 23S ribosomal RNA, uL22 and probably bL17 and bL32 proteins.
The interaction is driven by the C-terminal helix extension of the
PDF, which therefore acts as a master regulator of PDF-ribosome
binding [53]. The significance of this positively charged C-termi-
nal a-helix for binding to the ribosome and influencing cell viability
and growth rate raises the question of how the viral PDFs engender
their interaction with ribosomes. In a more general context, it as
well remains unclear how other PDF enzymes that do not belong to
the same class as the E. coli PDF interact with the ribosome. Indeed,
the C-terminal extensions of subtype 1A or 2 PDFs adopt different
folding compared to the C-terminal a-helix of subtype 1B [49]
(Fig. 2B). It should be also pointed out that PDFs of subtype 1B
and 1A or 2 can be present in the same bacteria or organelle
[45,54,55], triggering the question whether different types of PDFs
can simultaneously bind the same ribosome and more importantly
what are the physiological consequences for an organism to express
slightly different PDFs [49].
2.4. Methionine aminopeptidases
Methionine aminopeptidases (MetAPs) remove the initiator
methionine of newly synthesized proteins, which as a prerequisite
has to be deformylated by PDF. The MetAPs are essential in all
kingdoms of life, from bacteria to higher eukaryotes including yeast
and plants [11,56,57]. MetAPs are dinuclear metalloenzymes
evolutionary closed to creatinase, prolidase (DPP) and aminopep-
tidase P (APP) [11]. They all belong to the so called “pita-bread”
family or “clan MG” M24 protease family featuring a pseudo two-
fold axis of symmetry with the catalytic site and metal ion bind-
ing located at the interfaces between the domains (see legend of
Fig. 3 for more details) [56,58]. Metal requirement studies indicate
that MetAPs can be activated by various divalent metal cations but
unlike PDFs the physiological metals of MetAPs are still a matter of
debate [56,59]. As for PDFs, a low level of MetAPs sequence simi-
larity is compensated by similar 3D structures of the active site and
a conserved 5-residue metal binding site.
From sequence similarity and structural characterization two
types of MetAPs, MetAP1 and MetAP2, can be distinguished. Pro-
karyotes possess homologs of either MetAP1s (bacteria) or
furiosus). MetAP1a' from Streptococcus pneumoniae and human Ebp1 are also shown.
Structures are directed towards the substrate binding cleft of the catalytic site, with
catalytic residues of EcMetAP1a highlighted in magenta. The insertions within the
catalytic domain of type 2 MetAPs are highlighted in green and blue, and various N-
terminal extensions in red (the N-terminal polycharged domain of human MetAP2b is
partially visible in the X-ray structure). The binary complex formed by fungal Arx1 and
60S ribosomal subunit is shown, with the same color code for Arx1 compared to
MetAPs (bottom right).
138 C. Giglione et al. / Biochimie 114 (2015) 134e146
MetAP2s (archaea), while eukaryotes have both classes. The major
difference between MetAP1s and MetAP2s resides in the presence
of two additional compact sub-domains of unknown function
within the C-terminal catalytic domain of MetAP2s (Fig. 3A). The
absence or the presence of specific N-terminal extensions of both
prokaryotic and eukaryotic MetAPs further differentiates these
enzymes. Thus, MetAP1s can be classified into four subclasses
(Fig. 3). For example, E. coli possesses a MetAP1a displaying only the
catalytic domain [60] (Fig. 3), whereas other bacteria such as acti-
nobacteria display an additional MetAP1c characterized by an extra
N-terminal stretch of 40 residues bearing an SH3 binding P-X-X-P
motif that precedes the catalytic domain and is suggested to be
involved in binding to the ribosome [61] (Fig. 3). Since several ri-
bosomal proteins have SH3 domains and as uL24 is located adjacent
to the ribosomal exit tunnel, it has been suggested that MetAP1c
and the human type 1 MetAP might bind to this ribosomal protein
[61]. Recent findings have revealed that the N-terminal extension of
MtMetAP1c is indispensable for its activity likely by regulating
active site residues through a distance mechanism [62]. In the
cytoplasm of eukaryotes, on the other hand, MetAP1b contains two
domains composed by the catalytic domain followed by a 50-
residue linker and an N-terminal zinc finger domain proposed to
be responsible of interaction with the ribosome [63] (Fig. 3A). The
fourth subclass of MetAP1s is represented by the organelle-
targeted MetAP1d showing an N-terminal extension containing a
transient N-terminal signal peptide removed upon translocation of
the protein into the organelles [40] (Fig. 3). A genetic variant of
MetAP1s (MetAP1a'), present predominantly in Streptococci and
Lactobacilli and displaying a new insert in the catalytic domain (i.e.,
Streptococcus pneumoniae MetAP1a', Fig. 3B), has been recently
described [64]. This insertion is likely to undergo post-translational
modifications. Finally, MetAP2s are arranged in two subclasses
(MetAP2a and MetAP2b) with MetAP2b containing an extra N-
terminal domain composed by polybasic and polyacidic residue
blocks folding as an a-helix [65] (Fig. 3). One or two additional
insertions are present also in the catalytic domain of type 2 MetAPs
(Fig. 3).
The role(s) of each of the additional domains of the different
MetAP enzymes is still largely unknown. It is nevertheless clear
that they are not required for aminopeptidase activity [66] and,
considering their location with respect to the active site, they are
unlikely to explain the slight differences of substrate specificity of
MetAP types [67]. Interestingly, the additional domains protrude on
the very same side of the 3D structure (Fig. 3B). It is therefore likely
that these domains mediate interactions with various partners, and
contribute to regulate the NME process. Accordingly, it has been
shown that the first N-terminal polycharged Lys-rich block of
MetAP2b, previously called p67 [68], stores a POEP (protection of
eIF2a phosphorylation) activity [58], preventing the phosphoryla-
tion of the alpha subunit of eukaryotic initiation factor 2 (eIF2a) by
interacting with it [69e71]. When unphosphorylated, eIF2a can
bind to the ribosome and initiate translation, while its phosphor-
ylated form cannot bind, leading to inhibition of translation initi-
ation [58]. The whole process contributes to regulate the overall
rate of protein synthesis. It is still unclear how the two independent
activities, POEP and NME, are balanced taking into account the
extremely low level of the protein in the cell. Nonetheless, the
generation by autoproteolysis or calpain action of shorter MetAP2
molecules of about 50e57 kDa and 26 kDa, preserving an NME and
POEP activity respectively, might imply a separation of these two
activities at least in particular cellular conditions [72]. The ErbB-3
receptor binding protein (Ebp1), showing a conserved pita-bread
MetAP fold devoid of NME activity, appears to also have a POEP
activity situated at its C-terminal region (Fig. 3), allowing the in-
hibition of eIF2a phosphorylation in the cell [73,74]. Interestingly,
association of Ebp1 with mature ribosome has been also demon-
strated [74] and the crystal structure of the yeast Ebp1 homolog
Arx1 with the 60S ribosomal subunit has been recently solved
[75,76]. The binding of Arx1 has been mapped at the exit tunnel by
directly contacting (Fig. 3) several rRNA elements and ribosomal
proteins uL23, uL29 and uL24 [75e77]. Although the 3D map of
Arx1 bound to the 60S ribosomal subunit is not detailed, a never-
theless well-defined major contribution to the ribosome binding is
carried by the C-terminal catalytic domain insertion (Fig. 3B).
Hence, it was suggested (but not demonstrated) that MetAP2b
might bind to a ribosome in a similar manner and position as Arx1
[75]. An interaction between uL23 and bL17 of the ribosome and
bacterial E. coli MetAP1a has been also shown [78] and involves a
positively charged loop as a binding site for ribosomes (the putative
ribosome binding loop, Fig. 3B). Although no data is yet available
about how many distinct MetAP family members interact in a
productive manner with ribosomes and the nascent polypeptide
chains, the two above cited studies together with other evidence
seem to decline a common strategy that might be exploited by the
different MetAP family members.
3. Protein N-a-Acetylation
After NME is completed, N-a-Acetylation (NTA) is probably the
second most common protein modification as it affects more than
50% and 80% of yeast and human cytosolic proteins, respectively
[79e81]. Although protein acetylation is frequently associated with
histone and Lys modifications, protein NTA is distinct and co-
translationally targets the N-terminus amino group of the pro-
teins exclusively. This modification occurs also in archaea but with
a reduced frequency [82]. Available data indicate that bacterial NTA
also occurs on some proteins such as elongation factor Tu and some
ribosomal proteins but more rarely than in other organisms (Fig. 1).
NTA was the subject of recent large scale proteomic studies that
highlights the high frequency and the conservation of this process
even in organelles of eukaryotes [83]. Indeed, the presence of such
modification has been shown on the N-terminus of chloroplastic
proteins with an unexpected high frequency (more than 30% of the
plastid proteome). Since NTA occurs on nuclear-encoded chloro-
plastic proteins after the cleavage of a transit peptide, this N-ter-
minal acetylation appears to be post-translational despite it could
also be co-translational for the few chloroplast-encoded proteins as
well (Fig. 1). Less clear is the impact of NTA on mitochondrial
proteins to date, although the few data available on the mature N-
terminus of the nuclear encoded mitochondrial proteins seems to
suggest that this modification should be rare.
Although it has been proven that this modification is essential
for cell viability [84e89] and survival [90,91], very little is known
about the physiological reasons associated with this crucial role.
Since most accumulating cytoplasmic eukaryotic proteins are
terminally acetylated, NTA was considered to protect proteins from
degradation. Nonetheless, recent studies on specific protein targets
have revealed that one biological function of NTA is to create spe-
cific degrons inducing degradation of the protein by the protea-
some [28e30]. Involvement of NTA has been also shown to
influence membrane targeting, proteineprotein interactions
[92e94] and changes in proteostasis [95].
3.1. The N-a-acetyltransferase enzyme
NTA is catalyzed by N-a-acetyltransferase (Nat) complexes
generally composed in eukaryotes by a minimum of two sub-
units: a catalytic subunit accepting an acetyl-CoA as donor for
the transfer of the acetate moiety to the N-a-amino group
substrate and an auxiliary subunit (Table 1). In eukaryotes, 6
 C. Giglione et al. / Biochimie 114 (2015) 134e146 139

Table 1
Nats composition and their protein specificity. N-a-acetyltransferases (Nats) enzymes have been identified in all kingdoms of life. They contain at least one catalytic subunit,
which can be associated with one or two auxiliary subunit(s); names in parenthesis are for yeast proteins. No auxiliary subunits have been described for bacterial and archaeal
Nat. Naa10 and Naa15 are common to NatA and NatE, for which they play distinct roles. The substrate specificity is indicated for each class of Nats.

Eukaryotes Bacteria Archae

*
NatA NatB NatC NatD NatE NatF Rim Nat

Catalytic subunit Naa10 (Ard1) or Naa20 (Nat3) Naa30 (Mak3) Naa40 (Nat4) Naa50 (Nat5/San) Naa60 RimI/J/L NAT
Naa11a (Ard2) YhY
Auxilliary subunit(s) Naa15 (Nat1) or Naa25 (Mdm20) Naa35 (Mak10) d Naa10 d d d
Naa16a (Nat2) Naa38 (Mak31) Naa15
Substrates Ser- Met-Leu- Ser- Ser- / Ala-
Ala- Met-Asp- Met-Leu- Met-Leu- Met-Phe- Ala- Thr- / Gly-
Thr- Met-Glu- Met-Phe- Ser- Met-Phe- Met-Ile- Thr- Val- / Cys-
Gly- Met-Asn- Met-Ile- (H2A and Met-Ile- Met-Trp- Met-Leu-
Val- Met-Gln- Met-Trp- H4 histones) Met-Trp- Met-Lys- Met-Phe-
Cys- Met-Ile-
Met-Trp-
a
Naa11 and Naa16 are orthologues of Naa11 and Naa15 respectively, and are specific for higher eukaryotic NatAs. Each catalytic subunit of NatA (Naa10 and Naa11) can
therefore be associated to either one of the auxiliary subunits, leading to four different complexes. NatF has been identified only in higher eukaryotes.

distinct complexes have been identified so far (NatA, NatB, NatC,
NatD, NatE, NatF), each of them able to modify a specific cate-
gory of N-terminal substrates with some overlap for various Nats
differing also in subunit composition (Table 1) [for reviews see
Refs. [81,96,97]]. NatA, NatB and NatC are thought to be
responsible for the majority of NTA events. The NatA complex,
which is composed of the catalytic subunits Naa10 or Naa11 and
the auxiliary subunits Naa15 or Naa16, acetylates N-termini
exposed after removal of the first Met by NME (Fig. 1), mainly
Ser, Ala, Gly, Thr and Val (Table 1). Monomeric Naa10 has been
shown to also acetylate post-translationally mature actins
exposing Asp or Glu N-termini but not traditional NatA sub-
strates [98]. Moreover Naa10 seems to have also a lysine-specific
acetyltransferase KAT activity, transferring the acetyl group form
the Ac-CoA to the ε-amino groups of internal lysines of proteins,
and even an AT-independent activity, controlling the activity or
binding with other partners [for review see Ref. [97]]. NatB and
NatC are potentially NME-independent and acetylate proteins
retaining the initial Met when the second residue is acidic
(NatB) or hydrophobic (NatC) (Table 1). In eubacteria, RimI, RimJ,
RimL and YhY are believed to be homologs of eukaryotic Nats,
whereas only a single Nat complex with a broader substrate
specificity than eukaryotic NatA complexes seems to occur in
archaea [82,99,100] (Table 1). Although large proteomic analyses
of several plant species have shown substrate similarity to hu-
man Nats, limited information is available on plant NTA ma-
chinery [83,101e104].
To date, several Nats have been structurally characterized. This
includes 2 eukaryotic Nats, the Schizosaccharomyces pombe NatA
complex consisting of the Naa10 and Naa15 subunits, the human
NatE (the catalytic subunit Naa50), the unique archaeal Nat from
Sulfolobus solfataricus and several bacterial Nats [105e108]. The 3D
structures of all reported Nats place these enzymes in the Gcn5-
related N-acetyltransferase (GNAT) family, a large superfamily of
enzymes that use acyl-CoAs to acylate their substrates [109].
Members of the GNAT family are indeed characterized by a nearly
universally structurally conserved fold including an N-terminal
strand followed by two helices, three antiparallel b strands, a
central helix signature followed by a fourth a helix and a final b
strand [109]. The GNAT members use the same structural element
to serve as their oxyanion hole, i.e., the backbone amides in a
conserved “b bulge” located on strand b4 to perform acylation via
a variety of different chemical stategies involving sometimes
dramatic conformational changes. Differences between GNAT
structures are generally positioned at the N and C-termini [109]
(Fig. 4A). Moreover, the 3D structure of the active subunit of hu-
man NatE (Naa50) (Table 1) reveals crucial differences at the level
of the substrate-binding groove showing a more constricted
protein-substrate binding site allowing to the enzyme to accom-
modate only an a-amino substrate versus side chain Lys substrates
[106]. The structure of the S. pombe NatA complex shows that the
auxiliary Naa15 subunit is composed of 37 a-helices, among which
13 have conserved bundle tetratricopeptide repeat (TPR) motifs,
and wraps completely the Naa10 catalytic subunit. A number of
these TPR-helices are involved in Naa10 interaction and it has
been also suggested to favor interaction with other NatA binding
partners (Naa50 in NatE, see Table 1) or the ribosome [105].
Interestingly, the comparison of the 3D structures of fungal Naa10
shows a conformational change of the protein when it interacts
with Naa15, especially in the loop connecting a-helices 1 and 2
[105]. This suggests that the auxiliary subunit Naa15 induces an
allosteric conformational change of this Naa10 loop, leading to an
active conformation of the NatA catalytic subunit active site
required for the catalytic mechanism of the NatA complex
employing its traditional substrates [105]. Finally, the superim-
position of the Naa10 and Naa50 active sites revealed the impor-
tance of Naa10 Glu24 for excluding Glu and Met as amino-
terminal substrates and highlights the significance of Val29 in
human Naa50 for N-terminal Met recognition and acetylation
[105,106] (Fig. 4B). Interestingly, the 3D structure of the archaeal
ortholog, able to acetylate NatA and NatE substrates, presents the
acetyl-CoA binding core region and the flanking N and C-terminal
parts highly similar to the active form of Naa10 and Naa50.
However, it displays a non-conserved feature in the catalytic core
of the enzyme composed by an extended b3-b4 loop important to
stabilize the position of key residues such as His137 involved
particularly in Met amino terminal substrate acetylation [100].
Besides, there are several GNAT enzymes that take advantage of
the b3-b4 loop variability for functional gain [100]. It was there-
fore suggested that the archaeal Nat, by showing a larger substrate
specificity than eukaryotic orthologs, represents an ancestral Nat
variant.
3.2. Co-translational N-a-acetylation and its control
Several lines of evidence indicate that NTA can occur co- or post-
translationally [83,102,110e112]. Studies using synthetic substrates
and purified Nats have concluded that the protein substrate must
140 C. Giglione et al. / Biochimie 114 (2015) 134e146

Fig. 4. Similarities and differences between N-a-acetyltransferases (Nats) and N-myristoyltransferases (NMTs). (A) Both Nats and NMTs belong to the GNAT superfamily, which
members use acyl-CoAs to acylate their substrates. All members of GNAT superfamily display a common GNAT fold, consisting of an acyl-CoA binding site and a peptide binding site.
The GNAT fold is highly conserved around these ligand binding sites. Measurement of evolutionary conservation of amino acid positions in GNAT family members was achieved
with ConSurf 2010 [175,176]. The search for homologous sequences of Naa10 in its active form (PDB code 4KVX) was performed using PSI-BLAST against UniRef90 sequences
databank, the other parameters being the default ones. The continuous conservation scores are divided into a discrete scale of nine grades for visualization, from the most variable
positions (grade 1) colored turquoise, through intermediately conserved positions (grade 5) colored white, to the most conserved positions (grade 9) colored brown. These con-
servation scores are projected onto Naa10 structure represented as molecular surface. (B) Nats display the classical GNAT fold, with acetyl-CoA and peptide binding sites. Except for
NatD, the Nat substrate specificity lies within the first two to five residues, leading to a largely open peptide binding site (top inset). Residues contributing to the substrate specificity
are shown for Naa10 and Naa50 (middle and bottom insets). (C) The NMT fold is composed of two adjacent GNAT domains (highlighted in yellow and magenta), with the myristoyl-
CoA binding site carried by the N-terminal GNAT domain. The peptide binding site, recognizing the first eight amino acids, is shared by both the two GNAT domains (top and middle
insets). Residues involved in peptide binding are shown for human NMT1 (bottom inset).

contain a minimum of 10 and a maximum of 25 residues to be 4. N-myristoylation

acetylated [113,114]. Alternatively, in vivo investigations of yeast
and mouse NatA defined that association of the complex with a
ribosome is mediated by the auxiliary subunit Naa15 and required a
length of the nascent polypeptide emerging from the ribosome
(40e70 amino acids) longer than nascent chain necessary for
interaction with the nascent polypeptide-associated complex NAC,
an eukaryotic chaperone dimer, or the Ssb1p/Ssb2p proteins
forming a chaperone triad with the ribosome-associated complex
RAC [115e119]. This ribosome association seems not to be depen-
dent on the nature of the amino acid sequence of the nascent
polypeptide [116]. NatB, NatC and NatE have also been shown to
colocalize with ribosome and interaction between Naa15 and uL23
and uL29 has been highlighted [111].
Until recently, NTA was mostly considered as an irreversible
modification although some recent results [31,79,102,120] have
uncovered the occurrence of both the acetylated and non-
acetylated forms in the same sample of eukaryote species and
involving about 9% of all experimentally identified N-termini in
human cells [79]. Moreover, E. coli ribosomal protein L12/L7 is also
known to exist in two different forms i.e., N-acetylated and the
unacetylated counterpart. The total amount of both forms remains
constant but their ratio is dependent on the growth cycle [121]. No
N-alpha deacetylase is known so far but the characterization of
both modified and unmodified products change radically the idea
of a completeness of this modification. The origin of the unacety-
lated counterpart of a protein is yet to be ascertained. Several non-
exclusive hypotheses can be formulated such as: i) recurrent weak
identification of the N-terminal substrates by Nats (see above), ii)
regulation of Nat activity, e.g., availability of AcCoA has been shown
to control the degree of NTA of specific proteins [91] or/and iii)
regulation of ribosome Nat-binding.
NTA and the enzymes involved in this modification are currently
among the most challenging topics of molecular cell biology with
many open questions [96] which are expected to find an answer in
the near future.
N-terminal Myristoylation (MYR) is one of the most critical lipid
protein-modifications and consists in the addition of a saturated
C:14 fatty acid to a free N-terminal Gly residue of eukaryotic and
viral proteins exhibiting a favorable context at the level of the first 8
residues [122e125]. In rare cases, proteins from lower eukaryotic
parasites and bacteria can also undergo MYR by the eukaryotic host
cell [126]. An N-terminal Gly is exposed mostly as a consequence of
NME very early during protein biosynthesis (Fig. 1), although recent
studies indicate that such a modification can occur also later on a
newly exposed N-terminal Gly after a proteolytic cleavage medi-
ated by caspases during apoptosis or by removal of a leader peptide
from bacterial proteins imported into eukaryotic cells [10,127].
Thus, MYR as NTA can occur in a co- or a post-translational manner.
MYR is believed to cover a significant part of any eukaryotic
proteome, reaching 1e4% of all proteins in a given proteome [80]
(Fig. 1). Nonetheless, the exact composition of such a proteome
remains still problematic to identify because i) it is technically
difficult to evidence the lipidated proteins from homogenous bio-
logical samples, ii) the available experimental approaches aimed at
validating MYR from complex in vivo samples are limited as the
corresponding protein usually belongs to the low abundance
membrane proteome in any organism and iii) it is tricky to obtain
reliable MYR prediction with the bioinformatics tools available so
far [128]. Hence, only a very small subset of putative myristoylated
proteins has been experimentally checked through individual
studies [for review see discussion in Refs. [129]], while bioinfor-
matics prediction tools developed by various teams worldwide
indicate that the majority of myristoylated protein targets remain
to be confirmed [80,130e133].
4.1. N-myristoyltransferases
MYR is catalyzed by an acylase called myristoyl CoA:protein N-
myristoyltransferase (NMT), which transfers the myristoyl group
 C. Giglione et al. / Biochimie 114 (2015) 134e146
from a myristoyl-CoA onto the N-terminal Gly of the targeted
protein (Fig. 1). Higher eukaryotes share very similar N-myr-
istoylation enzyme systems, involving two NMTs (NMT1 and
NMT2) among which NMT1 is the most abundant enzyme
[123,125] and most efficient in vitro [129]. Lower eukaryotes display
only one NMT. NMT1 and NMT2 display a high level of sequence
identity with the major differences residing at the N-terminus of
the enzymes. As Nats, NMTs are monomeric enzymes belonging to
the GNAT superfamily [109]. Interestingly, the first 3D structures of
fungal NMTs revealed the presence of both an N-terminal and a C-
terminal GNAT domains, with low sequence homology to each
other, connected by a long loop [134,135] (Fig. 4C). Myristoyl-CoA is
bound to the N-terminal GNAT domain while the peptide is bound
to a substrate binding site shared by both GNAT domains (Fig. 4C).
Particularly, the N-terminal Gly of a substrate-peptide becomes
positioned towards the N-terminal GNAT domain, whereas the rest
of the recognized-peptide lies on a long groove situated on the C-
terminal GNAT domain (Fig. 4C). From structural and biochemical
investigation of the yeast and human NMTs it seems that these
enzymes follow an ordered biebi kinetic mechanism proceeding
through a myristoyl-CoA binding to the free enzyme, followed by
the peptide substrate, and the released of CoA before the myr-
istoylated protein [136e138]. The conserved oxyanion hole
involved during the catalytic mechanism, formed by the main chain
amide backbones of both F170 and L171, polarizes the carbonyl of
the thioester of the myristoyl-CoA and stabilizes the tetrahedral
intermediate generated after the binding of the peptide Glycine-
residue followed by nucleophilic attack by the amine and elimi-
nation of the thiol of CoA.
To date, it is assumed that all NMTs have common characteris-
tics but they are not completely redundant, suggesting distinctive
peptide substrate specificities [123,124,139e141]. Together with
the failure in finding a unique consensus sequence for the myr-
istoylable site, this implies that NMTs might adopt different cata-
lytic strategies, as already observed for other GNAT members
including Nats (see above) to accomplish substrate-specific MYR.
This would enable the enzyme to recognize several consensus
motifs with different catalytic efficiencies [80,129,142].
4.2. N-myristoyltransferase and translation
The N-terminus of NMTs is not required for catalysis [123,125]
and it was suggested that it might be involved in targeting the
enzyme to various subcellular environments such as the trans-
lational machinery [143]. Indeed, interaction of NMT with poly-
ribosomes has been evidenced in the past [144,145]. Since it was
observed that during protein biosynthesis the myristate was more
efficiently attached to a model myristoylated protein in the first
10 min of translation and considering an average rate of in vitro
polypeptide chain elongation for this protein of 10.5 residues per
min, it was suggested for all predicted myristoylated proteins that
this modification might occur co-translationally when the length of
the nascent chain is less than 100 amino acids [146]. Recently, it
was shown for both human NMT1 and 2 that the N-terminal basic
amino acid region called K box is sufficient and essential for binding
to the ribosome [147]. However, this K box is not conserved in other
NMTs, leaving open the question of which part of these NMTs is
involved in ribosome binding or even which region on the ribo-
some is concerned.
4.3. The physiological functions of myristoylation
Despite the poor knowledge on the whole pool of proteins un-
dergoing to MYR, it is already known that this modification is
linked with fundamental cancer-related [125] or pathogen-
141
response functions in animals and plants [126]. Together with
other proximal signals, the main role of this lipidation is to target
the protein to specific membrane compartments where the protein
will be an essential regulatory component of signal transduction
pathways involving many kinases or small G proteins. Due to its
function in cellular communication networks, it should be expected
that the myristoylated proteome is intrinsically dynamic to ensure
a proper cell communication. However, MYR has been considered
for long time an irreversible modification being necessary but not
sufficient to allow a protein to membrane anchoring [148,149]
suggesting that the dynamics of the proteome was mainly
ensured by the reversibility of the second signal. For several evi-
denced myristoylated proteins it has been shown that the second
signal can be provided by attachment of another lipid a palmitate,
or a polybasic motif or a domain that will interact with another
membrane-bound protein. Surprisingly, the dynamic scenario of
the myristoylated proteome has been revised by recent break-
through studies revealing for the first time the enzymatic removal
of the N-terminal moiety on specific proteins by the Shigella viru-
lence factor IpaJ [150] and that MYR alone localizes the proteins to
the endomembrane system with a partitioning between this
membrane compartment and the cytosol correlated with the cat-
alytic efficiency of the NMT [151,152]. In the latter case, it was
suggested that NMT and Nat complexes could compete at the level
of the ribosome for binding to specific nascent chains. This was
shown for two protein mutants, R3G-TNF and R3M-TNF, found to
become both N-acetylated and myristoylated and in an in vitro
translational system showing that the same subset of proteins
displays both modifications [153,154] Indeed, most available pre-
diction tools of these modifications, even if quite accurate, show
weaknesses in the prediction of NTA and MYR on N-Gly residues
[83], indicating that the two patterns might overlap and thus
compete for the corresponding modification enzymes when they
emerge from the exit tunnel of the ribosome.
5. Nascent polypeptide meeting ribosome-associated NPMs
and interplay with other RPBs
Translation requires intricate communication among multiple
components to achieve speed, accuracy, and regulation. Commu-
nication between ribosomes, translation factors, tRNA, mRNA, and
other cellular pathways is the mechanistic basis for the multiple
and tight levels of control that exist in translation. Recent findings
have revealed the functional importance of non-ribosomal proteins
that permanently or transiently associate with the ribosome.
Among non-ribosomal proteins, ribosome-associated protein
biogenesis factors (RPBs) are molecules acting as soon as a nascent
polypeptide reaches the exit from the ribosomal tunnel. Indeed, the
ribosome is a platform for co-translational events affecting the
nascent polypeptide, including protein folding, targeting to various
cellular compartments for integration into membrane or trans-
location, protein quality control and protein modifications. How the
RPBs involved in these processes act upon the nascent polypeptide
chain and how they dialog at the level of the exit tunnel turn out to
be one of the most exciting and challenging questions of the last
years. As described in this review and others [see for instance
[3,155]], the region around the exit tunnel seems to constitute a
general docking site for almost all investigated RPBs.
5.1. Ribosome as NPMs partner
Since early seventies, it was accepted that PDFs and MetAPs
were the first RPBs to interact with the nascent chain. Even before
cloning the first PDF gene, it was noticed that in bacteria significant
amount of deformylation always occurred within the first 5e8 s, a
142 C. Giglione et al. / Biochimie 114 (2015) 134e146
time that can increase to 10e20 s during starvation of one or more
amino acids [156]. Considering a constant elongation rate of 5
residues/sec in vivo, it was concluded that deformylation was taking
place when the length of the nascent chain was between 25 and
100 amino acids. In parallel, studies on specific bacterial nascent
chains have demonstrated that removal of the first formyl-Met was
occurring when the nascent chains where about 40e50 amino
acids long confirming previous results [78,157e159] (Fig. 5). Many
genes encoding PDFs and MetAPs with classical enzymatic activity
but displaying specific features have been discovered in most or-
ganisms and cellular compartments in the last fifteen years (see
above) and several of them have been shown to be present and act
simultaneously. Although most of these PDFs and MetAPs have
been extensively characterized enzymatically and structurally,
nothing is known about their behavior in respect to their rate of
deformylation and Met removal from the nascent chain in vivo, i.e.,
the length of the nascent chain when these processes are ensured
by each of them. No impact of the nature of the sequence has been
shown in vitro for several PDFs [160,161], whereas no data are
available to date on PDF nascent chain association properties
in vivo. This is true also for the different MetAP enzymes for which a
predominant dependency on the nature of the second residue and
of the 4e5 following residues has been shown in vitro [67]. None-
theless, using ribosome-nascent chain complexes comprising 40 N-
terminal residues of enolase fused to the SecM arrest sequence it
was shown that this nascent chain was not able to increase the
ribosome affinity for E. coli PDF1B and MetAP1b [78].
As discussed in the previous section, some Nat auxiliary sub-
units have been shown to interact with the ribosomal docking
protein uL23 and uL29 (Fig. 5) but no information to our knowledge
is available on NMT ribosomal binding proteins. Since the two
classes of enzymes display the same GNAT fold it is tempting to
speculate that NMTs will also lie on the ribosome in proximity of
the exit tunnel. Nonetheless, knowledge on how exactly NMTs and
Nats interact with the ribosome and other RPBs will shed light not
only in the mechanistic aspects of their interaction with ribosome
but also on their essential but not fully comprehended function(s).
5.2. Cooperation and competition between RPBs
In vitro biochemical studies have shown that E. coli PDF1B and
MetAP1b interact with ribosome-binding sites very close to each
other (around uL22, bL17 and bL32 for the first enzyme and bL17
and uL23 for the second one, Fig. 5) [53,78], raising the specula-
tion that in vivo they might influence each other's interaction on
the ribosome. Although not complete, an in vitro competition
between E. coli PDF1B and MetAP1b for binding to non-translating
or translating ribosomes has been evidenced [78]. However, it was
suggested that the extremely rapid kinetics of PDFs should allow
Fig. 5. Currently known ribosomal proteins involved in binding with NPM enzymes. This picture summarizes the whole set of different N-terminal modifications, the related
enzymatic factors (so-called RPB factors), the suggested nascent chain length when these modifications are expected to occur as well as the known ribosomal proteins interacting
with each RPB factors. The location of these ribosomal proteins is highlighted in bacterial and eukaryotic cytoplasmic ribosomes (right).
these enzymes to scan ribosomes in a very short time compared to
translation speed [37,49]. Although no similar kinetic constants
are available for MetAPs, same results were extrapolated for these
processing enzymes. For the moment, it remains a puzzle what is
the proportional weight of the respective independent contribu-
tion of PDF/MetAP-ribosome binding and the rapid PDF/MetAP
kinetics for an efficient and rapid NME process on a whole pro-
teome. Even more diffuse is our knowledge on the interplay be-
tween the NME enzymes and either bacterial or organellar
chaperone protein trigger factors (TF), for which the bacterial form
is known to interact with the bacterial ribosomal proteins uL23,
uL24 and uL29 [162e164]. After an initial hypothesis that PDF and
TF can act in a concerted fashion [53], it was later shown that
E. coli TF and PDF1B compete for ribosome binding in vivo [165],
i.e., TF overexpression slightly reduced cell growth, a phenotype
particularly evident when cells expressing endogenous E. coli PDF
were treated with the specific PDF inhibitor actinonin. This inhi-
bition by TF overexpression was even exacerbated when actinonin
inhibiting cells expressed the C-terminally truncated version of
E. coli PDF instead of the WT form [78]. Since it was previously
shown that the 21 last residues folding the C-terminal helical
extension of E. coli PDF is not necessary for ensuring the essential
role of PDF in vivo [161,166], it is uneasy to fully explain these data
to date, although interaction with the ribosome of the latter
extension was observed [53]. To even further complicate the sce-
nario several in vitro binding data show that TF and PDF or MetAP
can bind simultaneously to ribosomes or RNCs [78,167]. Particu-
larly, it has been proposed that TF remained bound when PDF
interacts with specific RNCs or non-translating ribosomes (only
partial or no changes of Kd values for binding of TF to ribosome
were observed in the presence of different PDF concentrations),
but in a modified arrangement [167]. The same results were re-
ported for E. coli MetAP. Recent in vitro studies have also consid-
ered the interplay at the level of the exit tunnel of the ribosome of
another RPB factor, the signal recognition particle (SRP) involved
in translocation of specific membrane proteins, with PDF or MetAP
and have revealed that the binding of PDF or MetAP to RNCs
doesn't influence the binding of SRP as already observed with TF
[167].
6. Conclusions-future perspective
The ribosome was proposed early on as a partner of NPM en-
zymes [see for instance as a pioneering work Ref. [168]]. However,
the inability to isolate these enzymes stably associated with ribo-
some preparations but being able to fully reproduce their activity
and specificity in vitro in the absence of ribosomes have led the
scientific community to neglecting the “ribosome contribution” on
these NPMs. During the recent years, several data have clearly
 C. Giglione et al. / Biochimie 114 (2015) 134e146
established that the ribosome is indeed an additional player at least
for some NPMs. Several alternative routes for involving an NPM are
nevertheless feasible, as NPMs appear to be added also post-
translationally without the ribosome as a required partner of the
respective enzymes involved. Thus, the exact role and the very
requirement of ribosome association need to be cautiously
addressed. Unambiguously however, physical interaction of NPMs
with ribosomes brings closer the nascent chain and the protein
modifiers. Such proximity is probably required to ensure full
modification of a protein without any possibility for it to escape the
modification prior to being completed.
It is important to mention that despite the organization of the
proteins around the exit tunnel is quite conserved in all studied ri-
bosomes, a much greater number of proteins is involved in the
eukaryotic ribosomal exit tunnel core itself (Fig. 5) and that the
eukaryotic “welcome committee” of proteins more loosely associ-
ated to the ribosome (including NPMs) is larger compared to its
prokaryotic counterpart. Nonetheless, the recent high resolution
structure of the large subunits of both yeast and mammal mito-
ribosome revealed several unique features including the region of
the exit tunnel making this complex divergent from other types of
ribosomes [169e171]. Moreover, differences have been noticed even
between the yeast and mammal mito-ribosomes and involving also
the previous identified exit tunnels (e.g., the conventional poly-
peptide exit site, PES, and the polypeptide accessible site, PAS). This
led to the proposal that they both are putative alternative interaction
sites for different classes of enzymes involved in the co-translational
events. However, the presence of a long mitochondria-specific
extension of uL23 in the PES of yeast suggested that PAS is the
only active protein channel in yeast mito-ribosomes. This seems not
to be the case for the mammal mito-ribosome leaving still open the
issue of the functionality of both PES and/or PAS [169,172]. Protein
expansion segments have been found on most of the ribosomal
proteins from cytosolic, chloroplastic or mitochondrial ribosome,
suggesting that this may be a prerequisite for keeping or even
improving efficient, timely and specific interactions with the
increased number of co-translational NPM enzymes.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by ANR projects Ribo-Dyn (ANR-2010-
BLAN-1510) and PalMyProt (ANR-2010-BLAN-1611) and grant ARC
SFI20111203841. The authors thank Dr Bruno Gronenborn for crit-
ical reading of the manuscript and Dr Adina Breiman (Tel Aviv
University) for strong support.
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