Molecular Process and Regulation of Protein

Translocation Across membrane in Eucaryote

As Assignment Take-Home Paper of Molecular Genetics

Lecturer : Prof. Ir. Triwibowo Yuwono, PhD

Digdo Sudigyo

17/419967/PMU/09178

STUDY PROGRAMME OF BIOTECHNOLOGY

GRADUATE SCHOOL

GADJAH MADA UNIVERSITY

YOGYAKARTA

2017
 Molecular Process and Regulation of Protein
Translocation Across Membrane in Eucaryote

I. Introduction
Subcellular compartmentalization is a distinctive feature of eukaryotic organisms.
As most of the protein content of the different organelles must be imported from the
cytosol, eukaryotic cells have developed specific systems for: (1) recognition of newly
synthesized polypeptides in the cytosol, (2) targeting of these proteins to their appropriate
organelle, (3) recognition of the substrate proteins by surface components of the
organelle, and (4) vectorial translocation of these proteins into or across the organelle
membranes (Schatz and Dobberstein, 1996). A combination of genetic and biochemical
approaches have produced a large body of information about translocation of precursor
proteins into the ER, mitochondria, and chloroplasts.
The systems that directly deliver the synthesized precursor proteins to the target
membrane, and that catalyze the integration into or the transfer across the membrane, are
structurally and functionally diverse. Nevertheless, a general scheme applies to these
pathways. First, the precursor proteins are synthesized in the cytoplasm and contain an
organelle-specific signal. This signal is both required and sufficient to drive the targeting
and the translocation process. Secondly, transport of the precursor proteins to the targeted
membrane occurs in an import-competent form, which involves the interaction with
cytosolic components. Thirdly, the precursor and/or the transporting proteins have to be
recognized by receptors at the organellar surface. Fourth, a translocation channel has to
be present in the target membrane that drives the transfer of the substrate protein across
the membrane or its integration into the membrane itself (Schnell and Hebert, 2003).
Despite these common principles the protein translocation systems can be further
classified by specific properties. For instance, two distinct principles exist with respect to
the folding state of the precursor protein. Here, precursor proteins can be translocated
either in a folded (known for nucleocytoplasmic, peroxisomal or TAT-dependent
translocation) or unfolded state (across the envelope membranes of mitochondria,
chloroplasts or the endoplasmic reticulum membrane). A further way to discriminate
between the translocation systems is the localization of the initial receptor complex. In

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 case of nuclear or peroxisomal translocation, the substrates are recognized by soluble
receptors, which themselves act as translocators. In all other systems membrane-inserted
receptors warrant recognition and specificity. The membrane-inserted receptor proteins
are classified into two categories. One set recognizes the “transporters” involved in
precursor protein delivery, e.g. chaperones, whereas receptors belonging to the other set
recognize the precursor protein itself (Wickner and Schekman, 2005).
II. Endoplasmic Reticulum Protein Import Pathways
In eukaryotic cells the majority of polypeptides destined to cross the ER
membrane are translated and translocated simultaneously. In addition to resident ER
proteins, many proteins destined for secretion or for residence in the plasma membrane,
the Golgi apparatus, lysosomes, and the endosomal compartments also enter the ER
lumen cotranslationally. Proteins targeted to the ER are synthesized with a signal
sequence usually in the amino-terminal region similar to the sequence described for
bacterial proteins utilizing the Sec pathway (Fig. 1). The SRP binds both to the signal
sequence in the nascent polypeptide and the translating ribosome and targets the complex
to the ER membrane (Fig. 1). Binding of the SRP to the translating ribosome results in
slowing of translation (Lauring et al., 1995).

Fig. 1. The signal recognition particle pathway of protein translocation into the
ER. The signal recognition particle (SRP) binds to its receptor (SR) in the ER
membrane as well as to the docked ribosome. The translocon consists of the
Sec61α,β,γ proteins, TRAM, and Sec63p. The translocation requires GTP
hydrolysis (+GTP). The protein synthetic machinery provides part of the driving
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 force to translocate the growing polypeptide chain across the membrane, and
protein synthesis requires both GTP and ATP. The Hsp70 GRP78 in the ER
lumen is required for the substrate to enter the ER lumen. The signal peptidase
(SPase) cleaves the hydrophobic signal peptide (SP) after the protein enters the
ER lumen.
The signal recognition particle consists of a complex of six polypeptides and one
molecule of RNA. The 54 kDa subunit of the SRP is responsible for binding to the signal
sequence, and this interaction increases the SRP’s affinity for GTP (Walter and Blobel,
1981; Krieg et al., 1986). The SRP binds to its receptor (SRαp/SRβp) on the surface of
the ER, and the ribosome binds to the translocation site on the ER membrane. The
interaction between the SRP and SRαp induces the hydrolysis of GTP. As a consequence,
the SRP is released into the cytosol, the ribosome binds to the ER membrane, and the
nascent polypeptide is transferred into the aqueous channel of the translocon.
The ribosome binds tightly to the Sec61p complex in the ER membrane. The
Sec61p complex consists of three polypeptides, Sec61αp, Sec61βp, and Sec61γp (Fig. 1).
This protein complex is responsible for the formation of the aqueous channel and the
initial recognition of the signal sequence during the insertion of the nascent polypeptide
into the translocon. Interestingly, Sec61αp and Sec61γp are the eukaryotic homologues of
bacterial SecYp and SecEp, respectively. The translocating chain-associated membrane
(TRAM) protein preferentially interacts with the signal sequence of most of the proteins
translocated during the early stages of protein translocation but its exact function in
protein translocation is not clear. The SR, the Sec61p complex, and the TRAM protein
constitute the minimal requirement for reconstitution of protein translocation in
liposomes (Gorlich and Rapoport, 1993).
All eukaryotic cells also have an SRP-independent pathway where fully translated
ER precursor proteins are targeted to and translocated across the ER membrane. The
hydrophobicity of the signal sequence of the ER precursor may determine whether a
polypeptide is targeted by the cotranslational or the posttranslational pathway. Precursor
proteins with very hydrophobic signal sequences are preferentially recognized by the
SRP, while less hydrophobic signal sequences could follow either pathway (Ng et al.,
1996).

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III. Mitochondrial Protein Translocation
Although mitochondria contain their own genome, most mitochondrial proteins
are encoded by nuclear genes and are synthesized on free ribosomes in the cytosol as
precursors. Mitochondrial biogenesis requires protein targeting to four compartments: the
outer membrane, the intermembrane space, the inner membrane, and the matrix (Fig. 2).

Fig. 2. Translocation of proteins into mitochondria. Mitochondria have four different

compartments: the outer membrane (OM), inner membrane (IM), intermembrane space
(IMS), and the matrix (upper left). Protein precursors are bound by a complex of
molecular chaperones, and, in the presence of ATP, maintain the precursors in a
transport-competent state. Tom20/22 and Tom37/70 bind to different substrate proteins.
Tom5/6/7 modulate the translocation of substrate proteins across the translocon formed
by Tom40. Proteins destined for the mitochondrial matrix are translocated across the
inner mitochondrial membrane through a translocon formed by Tim23 and Tim17.
Translocation requires a proton-motive force (ΔpH/ΔΨ) across the inner membrane.
Tim44 interacts with Tim23 and a mitochondrial heat shock protein of 70 kDa (mtHsp70)

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that hydrolyzes ATP and pulls the substrate protein into the mitochondrial matrix. The
mitochondrial processing protease (MPP) cleaves the signal peptide (SP) from the
substrate. Proteins that remain in the matrix will be refolded with the help of mtHsp70
and the chaperonin 60 kDa molecular chaperone. Proteins destined for residence in the
inner membrane may first enter the matrix and then return to the inner membrane using a
second signal sequence exposed after the first has been removed (not shown).
Alternatively, inner membrane proteins may be directly targeted in a pathway requiring
Tim9/10 and Tim8/13 in the intermembrane space. The inner membrane translocon used
in this case is a complex of Tim22 and Tim54.
Translocation of mitochondrial precursors is an energy-dependent process that is
assisted by heteromeric translocation complexes in both membranes. Four translocation
complexes have been identified, two translocons in the outer mitochondrial membrane
(Tom) and two in the inner mitochondrial membrane (Tim) (Fig. 2). Many matrix
proteins cross the outer and inner mitochondrial membranes at locations of close contact
between the two membranes. Interestingly, Tim23 spans both the inner and the outer
membranes likely at contact sites. In addition, some proteins can be imported into
mitochondria independently of these translocation complexes perhaps due to their
abilities to spontaneously associate with membrane bilayers or to associate with the
translocon without the aid of receptors (Donzeau et al., 2000).
Precursor proteins destined to the mitochondrial matrix are usually hydrophilic
polypeptides with an amino-terminal signal peptide that forms amphipathic helices in
solution. On the other hand, inner membrane proteins, such as the ATP/ADP carrier
(AAC) and the phosphate carrier, are hydrophobic and contain internal targeting signals
yet to be determined. Both types of precursor proteins share the general import pore
(GIP) for transport across the mitochondrial outer membrane, but they bind to a distinct
set of receptors. The GIP complex is defined by the pore-forming protein Tom40 and the
small integral membrane proteins Tom5, Tom6, and Tom7 (Truscott, and Pfanner, 1999)
(Fig. 2).
Mitochondrial precursor proteins are imported in an unfolded state. Cytosolic
molecular chaperones such as Hsp70s and the mitochondrial stimulating factor (MSF)
maintain the newly synthesized precursor proteins in a transport-competent state and

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target them to mitochondria. MSF is an ATP-dependent cytosolic chaperone that interacts
preferentially with certain mitochondrial precursors (Hachiya et al., 1994). Other
cytosolic chaperones are likely to be involved in this translocation pathway since
mitochondrial precursor proteins exist as high molecular weight complexes.
Matrix precursors are recognized by the membrane receptors Tom20 and Tom22,
the cytosolic domains of which are capable of recognizing different characteristics of the
signal peptide. After binding to the receptors, the precursor protein interacts with Tom5, a
protein thought to be involved in transferring the precursor from the receptors to Tom40,
the main component of the translocation channel. The transition from receptor-bound
state to insertion into the GIP is ATP-dependent (Pfanner and Neupert, 1987) (Fig. 2).
Tom6 regulates the activity of Tom22 (Dekker et al., 1998). The driving force for protein
translocation across the outer membrane has yet to be determined. The sequential binding
of the positively charged signal sequence with increasing affinity to acidic domains of the
GIP proteins would allow the translocation of the signal sequence. An acidic domain has
also been identified in the intermembrane domain of the inner membrane protein Tim23,
the protein that actually spans both the inner and the outer membranes (Donzeau et al.,
2000).
The signal sequence of matrix precursor proteins first interacts with the
intermembrane space domain of the inner membrane protein Tim23, which together with
Tim17 forms the translocation pore complex (Fig. 2). Translocation across the inner
membrane is dependent on the proton-motive force. Bauer et al.,1996, have shown that
Tim23 can also regulate the opening of the pore. A third protein, Tim44, is also part of
the inner membrane translocation process. Tim44 is a peripheral protein that binds to the
intralumenal domain of Tim23 (Fig. 2). Tim44 acts as the docking protein for the matrix
chaperone mitochondrial Hsp70 (mtHsp70) and contains a DnaJ-like domain. mtHsp70 is
involved in both translocation and refolding of the translocated proteins. These
interactions are ATP-dependent. Disruption of the Tim44–mtHsp70 interaction has
shown that binding of mtHsp70 to the preprotein is not enough to promote translocation
of polypeptides that are not fully unfolded (Voos et al.,1996). mtHsp70, when anchored
to the membrane, may be capable of undergoing conformational changes induced by ATP
binding and hydrolysis that result in the ‘pulling’ of the precursor protein into the matrix.

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 The removal of the signal sequence takes place in the matrix at an early import
stage (Fig. 2). The major signal protease is the matrix processing peptidase (MPP), a
heterodimer formed by α- and β-subunits (Fig. 2). Removal of the signal sequence is
essential for the proper folding and function of matrix proteins. Proteins destined to the
inner mitochondrial membrane can first be targeted to the matrix and then imported into
the inner membrane. These proteins usually bind to the cytosolic face of Tom20, one of
the receptors for matrix preproteins. However, other inner membrane proteins bind to the
surface receptor Tom70/Tom37 (Dietmeier et al., 1997) (Fig. 2). The existence of
secondary or alternative receptors for mitochondrial protein translocation explains the
observation that deletion of neither Tom20 nor Tom70 is lethal. Similarly to
mitochondrial matrix precursor proteins, the inner membrane preprotein interacts with
Tom5, part of the GIP complex. During translocation across the outer membrane, the
protein substrate interacts with both Tom40 and Tom22 which form the aqueous pore.
When the inner membrane protein emerges in the intermembrane space it associates with
the intermembrane space complex formed by Tim9 and Tim10. The Tim9/10 complex
seems to be responsible for driving the protein across the GIP into the intermembrane
space (Adam et al., 1999). In addition, two other soluble proteins in the intermembrane
space, Tim13 and Tim8, have homology to Tim9 and Tim10 respectively, and they might
associate with Tim9/10, but their function is not known (Koehler et al., 1999). Tim12 is a
peripheral inner membrane protein facing the intermembrane space, and it is tightly
associated with the Tim22/Tim54 complex (Fig. 2).
Two interesting exceptions to the pathways described above are followed by the
inner membrane proteins Tim22 and Tim54 that together participate in the insertion of
inner membrane proteins. Tim22 binds to the surface receptor Tom20, instead of Tom70,
and then is inserted through the Tim22/Tim54 complex. On the other hand, Tim54 binds
to Tom70 and it is inserted into the inner membrane through the Tim23/Tim17 complex
(Kurz et al., 1999).
Outer membrane precursor proteins, such as porins, are also inserted through the
GIP. Tom7 destabilizes the interactions between Tom40 and Tom22, and this effect
allows for lateral movement and the insertion of the transmembrane protein into the lipid
bilayer of the outer membrane. Tom40 also uses the GIP complex for import into the

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 outer membrane and integrates into preexisting Tom complexes. Interestingly, Tom40
seems to enter the translocation pore partially folded as a requirement for proper insertion
into the outer membrane (Rapaport and Neupert, 1999). Protein residents of the
intermembrane space, such as Tim8, Tim9, Tim10, and Tim13, are also transported
through the GIP complex but they do not require the receptor proteins Tom22/Tom20.
Instead, Tom5 is required for translocation of these proteins (Kurz et al., 1999) (Fig. 2).
IV. Chloroplast Protein Translocation Pathways
Chloroplasts have at least six distinct compartments: outer membrane,
intermembrane space, inner membrane, stroma, thylakoid membrane, and thylakoid
lumen (Fig. 3). Each compartment contains specific proteins. For example, the thylakoid
membranes within the chloroplast contain the proteins responsible for photosynthesis and
electron transport. Chloroplasts, like mitochondria, contain their own genome, but most
chloroplast proteins are synthesized on cytosolic ribosomes and are imported
posttranslationally. These proteins are synthesized with targeting sequences that are
subsequently cleaved from the proteins after import. These targeting sequences are highly
variable in length (from 20 to more than 120 amino acids) but contain basic amino acids
and a high content of serine and threonine (Chen and Schnell, 1999; Schnell, 1995).
These targeting sequences do not fold into secondary or tertiary structures in an aqueous
environment, but form amphipathic β-strands or α-helices in a hydrophobic environment
(May and Soll, 1999).

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.
Fig. 3. Translocation of proteins into chloroplasts. Chloroplasts contain six different
compartments: outer membrane (OM), inner membrane (IM), intermembrane space
(IMS), stroma (STR), thylakoid membrane (M), and thylakoid lumen (L) (upper right).
Substrate proteins contain signal sequences that are recognized by molecular chaperones
tightly associated with the chloroplast outer membrane (Com70). The actions of Com70
as well as other aspects of the translocation process are stimulated by ATP (+ATP).
Association of substrate proteins with the Toc159/75 translocon is stimulated in the
presence of GTP (+GTP). An hsp70 at the inner surface of the outer membrane aids in
the transfer of substrate proteins to the Tic complex. A chloroplast hsp70 in the stroma
helps the precursor protein transit the membranes and also helps to refold proteins in the
stroma. A signal peptidase (SPase) cleaves the signal peptide (SP). Other molecular
chaperones including Clpc and Cpn60 may contribute to import of substrate proteins and
to their folding in the stroma. Proteins can enter the thylakoid lumen or membrane by a
variety of mechanisms related to the Sec pathway, the TAT pathway (ΔpH/ΔΨ), the SRP
pathway. cSecYEp, chloroplast homologues of SecYEp.
Protein translocation across the outer and inner membrane occurs simultaneously
for most proteins, probably at regions where the outer and inner membranes are in close

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contact. The translocon at the outer membrane of chloroplasts (Toc) binds to a substrate
protein and transfers the protein to the translocon at the inner chloroplast membrane
(Tic). This process is greatly stimulated by ATP (Fig. 3). Without ATP, only weak
binding of substrate proteins to Toc159 and Toc75 occurs (Chen and Schnell, 1999). In
addition to being receptors, Toc75 and Toc159 form the aqueous pore through which the
precursor protein translocates (May and Soll, 1999). The diameter of this pore is only 8–9
Å suggesting that proteins must be fully unfolded to translocate into chloroplasts (Chen
and Schnell, 1999). Toc159 and Toc34 also bind GTP and have intrinsic GTPase
activities (Fig. 3). Toc36 is required for optimal rates of protein translocation, but its
mechanisms of action are not yet known. The chloroplast outer membrane protein of 70
kDa (Com70) is an Hsp70 family member that is tightly associated with the outer
membrane (Kourtz and Ko, 1997) (Fig. 3). It binds to substrate proteins at an early stage
of translocation and is an especially active protein unfoldase in the presence of ATP (Fig.
3). Another Hsp70 family member is associated with the inner face of the outer
membrane and plays a role in delivery of substrate proteins from the Toc complex to the
Tic complex (May and Soll, 1999; Chen and Schnell, 1999 ) (Fig. 3).
Tic is composed of Tic110, Tic22, and Tic20 (Fig. 3). Tic110 can bind to
precursor proteins, and the proteins translocate across the inner membrane in close
association with Tic20 and Tic22 (Wu et al., 1994). Tic110, Tic20, and Tic22 do not
form a stable association except when interacting with the Toc complex. Insertion of the
precursor protein into the Tic complex requires ATP hydrolysis within the stroma (Chen
and Schnell, 1999) (Fig. 3). Interestingly, Tic22 enters the intermembrane space of the
chloroplast by a mechanism completely different than that described for stromal proteins.
Tic22 does not compete for chloroplast translocation with any other precursor protein
examined. Whether or not other intermembrane space chloroplast proteins follow this
unique translocation pathway remains to be established (Kouranov et al., 1999).
When the precursor protein reaches the stroma, a signal peptidase removes the
precursor sequence (Chen and Schnell, 1999). The molecular chaperones Clpc and Cpn60
associate with Tic110. Clpc and the chloroplast Hsp70 (cHsp70) may be required for
protein transport across the inner membrane, while Cpn60 assists in protein refolding in
the stroma (May and Soll, 1999; Chen and Schnell, 1999).

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 Many proteins including ferredoxin and ribulose 1,5-bisphosphate carboxylase
reside in the chloroplast stroma after import, but others enter the thylakoid membrane or
lumen. Some proteins including the light harvesting chlorophyll-binding protein enter the
thylakoid lumen using components homologous to the Sec apparatus. These proteins
often have a second targeting sequence that is exposed at the amino terminus after the
initial precursor has been cleaved by the stromal signal peptidase (Fig. 3). Proteins
containing very hydrophobic thylakoid targeting sequences also require a stromal
homologue of SRP. The import of both of these signal containing proteins is by SecY
(Laidler et al., 1995) and SecE (Schuenemann et al., 1999) homologues in the thylakoid
membrane and a SecA homologue (Nakai et al., 1994; Yuan et al., 1994) in the
chloroplast stroma. The translocation requires ATP hydrolysis (Fig. 3). Other proteins are
imported into the thylakoid lumen by a pathway similar to the TAT pathway for export of
bacterial proteins. These proteins are transported in a folded state, and they contain a twin
arginine motif in their targeting sequences (Thompson et al., 1999). The pH of the
chloroplast stroma is 8.0 while that of the thylakoid lumen is 5.0, and the TAT
translocation process is dependent upon the proton-motive force. There is also evidence
for a ΔpH-dependent translocon in the thylakoid membrane. Finally, several proteins
involved in photosynthesis and electron transport insert into the thylakoid membrane by
mechanisms that are independent of the Sec, SRP, or the TAT pathways (Fig. 3). Some of
these proteins may require Albino3, a homologue of bacterial YidC, localized within
chloroplasts (Sundberg et al., 1997).
V. Peroxisomal Protein Import Pathways
Peroxisomes are surrounded by a single membrane, and some of the proteins
within that membrane may be targeted to peroxisomes through the ER and Golgi by
means of vesicular traffic. However, most peroxisomal membrane proteins are
synthesized in the cytosol and inserted into the membrane posttranslationally. This latter
pathway requires initial binding of the protein to another peroxisomal membrane protein
prior to insertion into the lipid bilayer. The insertion is temperature-dependent and
requires ATP for certain peroxisomal membrane proteins but not others. The mechanisms
of import of peroxisomal membrane proteins remain an important gap in our
understanding of protein translocation (Terlecky and Fransen, 2000).

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 Peroxisomal matrix precursor proteins are synthesized in the cytosol and then
imported into peroxisomes (Lazarow and Fujiki, 1985). Peroxisomal proteins are
designated by a unified nomenclature as ‘peroxins’, and the genes involved in
peroxisomal biogenesis are represented by the acronym, PEX (Distel et al., 1996).
Proteins can be translocated into the peroxisomal matrix in a folded state (McNew and
Goodman, 1994), a feature shared with the bacterial TAT translocation pathway and its
homologous pathway for translocation of proteins into or across the thylakoid membrane
of chloroplasts.
Genetic and biochemical studies indicate that two classes of signal sequences are
responsible for targeting of peroxisomal matrix precursors (Fig. 4). The peroxisome
targeting signal 1 (PTS-1) is the most common signal peptide in proteins destined to the
peroxisome lumen (Subramani, 1998). PTS-1 is a tripeptide present at the carboxyl
terminus of the polypeptide, and it is loosely based on the sequence serine-lysine-leucine
(SKL) (Gould et al., 1996). Further studies have shown that alanine or cysteine can
substitute for serine, arginine and histidine can substitute for lysine, and methionine can
replace leucine, so the PTS-1 is more accurately described as S/A/C-K/R/H-L/M
(Subramani et al., 2000). The PTS-1 targeting peptide is not removed from the protein
after import into the peroxisome lumen (Fig. 4).

Fig. 4. Translocation of proteins into peroxisomes. Substrate proteins contain PTS-1 or

PTS-2 targeting sequences. Different cytosolic receptors, Pex5p and Pex7p, recognize

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these sequences and direct the proteins to peroxisome receptors, Pex17/14p or Pex14p
and translocons, Pex10/12/13p. Hsp70s and ATP (+ATP) stimulate translocation. The
PTS-1 receptor, Pex5p, enters the peroxisome along with substrate proteins, and proteins
cross the membrane in a folded state. The Pex5p is recycled to the cytosol. No role for
molecular chaperones in the lumen of the peroxisome has yet been proved.
The peroxisomal targeting sequence 2 (PTS-2) is found near the amino terminus
of polypeptides such as 3-keto-acyl-coenzyme A thiolase and consists of a peptide of
nine amino acids with the consensus sequence arginine/lysine-leucine/isoleucine/valine-
x5-histidine/glutamine-leucine/alanine (R/K-L/I/V-X-X-X-X-X-H/Q-L/A) (Subramani,
1998; Terlecky and Fransen, 2000). PTS-2 targeting sequences are active when
engineered to be at locations within the protein sequence other than the amino terminus.
PTS-2 targeting signal sequences are removed after entry into the peroxisome by a signal
peptidase in plants and mammals but not in yeast (Terlecky and Fransen, 2000).
Targeting of integral peroxisomal membrane proteins mentioned earlier is independent of
both PTS-1 and PTS-2 pathways (Chang et al, 1999).
The peroxisomal protein Pex5p has been identified as the receptor for the PTS-1
signal sequence (Dodt and Gould, 1996; Terlecky et al., 1996) (Fig. 4). Upon binding of
the receptor to the substrate protein, the receptor–substrate protein complex is targeted
from the cytosol to the peroxisomal membrane where Pex5p binds to the integral
peroxisomal membrane protein Pex13p (Erdmann and Blobel, 1996.) and/or the
membrane protein Pex14p (Albertini et al, 1997; Fransen et al, 1998; Girzalsky et al,
1999) (Fig. 4). The proteins that actually form the translocon in peroxisomal membranes
have not been conclusively identified. However, Pex13p in the peroxisomal membrane
appears to be in a complex with Pex12p and Pex10p, and deletions of Pex12p or Pex10p
block protein import at steps subsequent to substrate binding (Kalish et al, 1996; Chang
and Gould, 1998). The peripheral membrane protein Pex17p binds to Pex14 (Girzalsky et
al, 1999) and has also been implicated in the Pex5p receptor binding. Therefore, we
speculate that the peroxisome translocon may be formed by Pex10/12/13/17 (Fig. 4).
Pex5p actually enters the peroxisomal lumen with PTS-1 substrate proteins (Fig.
4). However, the frequency with which Pex5p enters the peroxisome is not known.
Interestingly, Pex4p is a peroxisomal membrane protein that may participate in the

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recycling of the Pex5p receptor back into the cytosol since the ΔPEX4 deletion results in
Pex5p accumulating within the peroxisome lumen (van der Klei et al., 1998). This entry
of the receptor along with the substrate protein into the organelle may also apply to the
lysosomal uptake of proteins (see below).
Pex7p, the PTS-2 receptor, is mostly localized to the peroxisomal membrane and
specifically binds to PTS-2 signal sequences and targets the precursors to the peroxisome
membrane (Marzioch et al., 1994; Zhang and Lazarow, 1996). Pex7p is able to bind to
the peripheral membrane protein Pex14p which interacts with the integral membrane
protein Pex13p. Therefore, Pex14p seems to be the site of convergence of both PTS-1
and PTS-2 pathways. Both PTS-1 and PTS-2 targeted peroxisomal proteins share the core
translocon consisting of Pex10/12/13p (Fig. 4). It is not yet known whether or not Pex7p,
like Pex5p, enters the peroxisome along with substrate proteins.
Both Hsp70 and the heat shock cognate protein of 70 kDa (Hsc70) are associated
with the cytosolic side of the membrane of purified rat liver peroxisomes (Fig. 4).
Microinjection of anti-Hsc70 antibodies into intact cells, or depletion of Hsc70 from
cytosol added in a permeabilized cell assay significantly inhibited peroxisomal transport
(Walton et al., 1994; Crookes and Olsen, 1998). Members of the Hsp70 family have also
been observed in the lumen of plant peroxisomes (glyoxisomes) (Corpas and Trelease,
1997; Diefenbach and Kindl, 2000). In addition, a plant Hsp70 interacts with a
peroxisomal membrane-anchored DnaJ/Hsp40 homologue. Further fractionation of
purified peroxisomes from cucumber cotyledons indicates that two isoforms of Hsp70
and a soluble form of the Dna-J/Hsp40 homologue were present in the lumen of
peroxisomes. On the other hand, no Hsp70 protein was found in the peroxisomal lumen
from rat liver peroxisomes. These contradictory results could be explained by actual
differences between the mammalian and the plant systems or by differences in the
sensitivity of the techniques and reagents used for the immunodetection. A requirement
for molecular chaperones in the targeting of matrix proteins to peroxisomes is somewhat
surprising since protein unfolding is not required for this protein translocation pathway
(McNew and Goodman, 1994). Perhaps the requirement for molecular chaperones is for
their role in facilitating assembly of protein complexes.

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VI. Protein Translocation Into Lysosomes
Lysosomes are able to take up and degrade proteins by several pathways involving
vesicular traffic (Dice, 2003). Exogenous proteins as well as membrane proteins can be
delivered to lysosomes by endocytosis (D’Hondt et al., 2000). Secretory proteins can be
delivered to lysosomes for degradation when the secretory vesicle fuses with a lysosomal
membrane instead of the plasma membrane. This process, crinophagy, is often activated
when the demand for the secreted protein is low (Lenk et al., 1991). For example,
crinophagy of insulin by the insulin producing β-cells increases when blood glucose
levels are low. Under these circumstances demand for insulin secretion is low. Cytosolic
and organelle proteins can be taken up by lysosomes by the processes of macroautophagy
and microautophagy. In macroautophagy, regions of cytoplasm are first surrounded by a
double membrane to form an autophagosome, and many of the mechanisms responsible
for this process have been recently discovered (Kim and Klionsky, 2000; Klionsky and
Emr, 2000). The autophagosome acidifies and then fuses with lysosomes.
Microautophagy refers to the indentation of the lysosomal membrane to form tubules
which then pinch off to yield vesicles within the lysosome (Muller et al., 2000; Sattler
and Mayer, 2000). The membrane of such vesicles breaks down to release internalized
materials into the lysosomal lumen.
In addition to these vesicular pathways, lysosomes are able to take up cytosolic proteins
for degradation in a molecule-by-molecule fashion (Dice, 2000; Cuervo and Dice, 1998).
This process, chaperone-mediated autophagy, is activated by prolonged starvation or by
the removal of serum growth factors from confluent cells in culture (Cuervo et al., 1995
and 2000; Neff et al., 1981). This pathway of proteolysis is similar to the other protein
translocation systems discussed in this review. Substrate proteins contain targeting
sequences related to lysine-phenylalanine-glutamate-arginine-glutamine (KFERQ) (), and
Hsc70 and cochaperones stimulate this proteolytic pathway (Backer et al., 1983 and
1986; Chiang and Dice, 1988) (Fig. 5). At least one role of the molecular chaperone is to
unfold protein substrates. Dihydrofolate reductase (DHFR) is a substrate for chaperone-
mediated autophagy, and methotrexate is known to stabilize the conformation of DHFR.
Methotrexate markedly inhibits transport of DHFR into lysosomes but does not affect its

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binding to the lysosome surface (Salvador et al., 2000). These results suggest that
proteins must be unfolded to translocate across the lysosomal membrane.

Fig. 5. Protein translocation into lysosomes. Substrate proteins contain a targeting peptide
that is recognized in an ATP-dependent manner by a molecular chaperone complex
including Hsc70. This complex of chaperones is also associated with the lamp2a in the
lysosomal membrane. An hsp70 in the lysosomal lumen (lyHsc70) is required to pull the
substrates into the lysosome, but a requirement for ATP has not yet been shown. A
requirement for a proton-motive force in protein translocation is unknown (ΔpH/ΔΨ ?).
Soon after import the protein is degraded by the high concentrations of cathepsins in the
lysosome.
Molecular chaperones within the lysosomal lumen are required for the import of
substrate proteins (Agarraberes et al., 1997; Cuervo et al., 1997) (Fig. 5). Most substrate
proteins bind to a receptor at the lysosomal surface, the lysosome-associated membrane
protein 2a (lamp2a) (Cuervo and Dice, 1996). Lamp2a levels in the lysosomal membrane
are dynamically regulated (Cuervo and Dice, 20001), and the level of lamp2a directly
correlates with the activity of the pathway under a wide variety of physiological and
pathological conditions (Cuervo and Dice, 20002).

16
The translocon in the lysosomal membrane has not yet been conclusively identified, but it
may be that the lamp2a receptor also forms the protein translocation channel through the
membrane. Lamp2a multimerizes into tetramers, octamers, and larger homomultimers
(Cuervo and Dice, 20002) and so could provide multiple membrane spanning protein
segments.
Have identified two variants of Hsc70 that differ in their pI and location in the
lysosome (Agarraberes and Dice, 2001). The most acidic variant (pI=5.3) is localized
within the lysosomal matrix (lyHsc70) while the less acidic form (pI=5.5) is associated
with the cytosolic face of the lysosomal membrane (lymHsc70). Both variants of Hsc70
are required for transport of substrate proteins (Agarraberes et al., 1997). We are
characterizing several other polypeptides that form a large molecular weight complex
with lymHsc70 (Fig. 5).
This complex could participate in maintaining a tight seal of the translocation
complex in a similar manner as described for the translating ribosome bound to the
Sec61p complex in the ER on the cytosolic side of the membrane (Zimmermann, 1998).
At the same time it might act as a receptor for certain substrates and that might not
require additional stable binding to lamp2a. Finally, this complex at the lysosomal
membrane is likely to be responsible for the unfolding of protein substrates known to be
required for their transport into lysosomes by this pathway (Salvador et al., 2000).
Cuervo and Dice have recently determined that a fraction of lamp2a is in the lumen of the
lysosome forming a lipid–protein complex perhaps with cholesterol (Cuervo and Dice,
20001). They proposed a dynamic model where the lumenal lamp2a can be recruited to
the lysosomal membrane upon activation of chaperone-mediated autophagy. It is possible
that cholesterol micelles containing lamp2a intercalate into the lysosomal membrane and
then lamp2a incorporates into preexisting translocation complexes by lateral diffusion.
This model would explain the fact that upon activation of chaperone-mediated autophagy
the levels of lamp2a increase (Terlecky and Dice, 1993; Cuervo and Dice, 20002). The
concentration dependence of multimerization of a transmembrane protein may not be
linear so that the amount of octamers may increase dramatically in response to a 2-fold
increase in lamp2a in the lysosomal membrane.

17
 The interaction of protein substrates with lamp2a most likely positions these substrates
for insertion into the translocation pore. A similar interaction has been proposed between
Tom5 and preproteins on the outer membrane of the mitochondrion (Truscott and
Pfanner, 1999) and for Toc159 on the outer membrane of chloroplasts (Chen and Schnell,
1999).
VII. Conclussion
The different protein translocation systems reviewed have several common. Many
translocation pathways require ATP and/or GTP. A notable exception to this requirement
is the evolutionarily conserved pathway into or across the chloroplast thylakoid
membrane. A requirement for a proton-motive force is also common but not universal.
Alternative driving forces must exist in many translocation systems. A role for molecular
chaperones in the cytosol, in the organelle lumen, and even in the organelle membrane is
a common feature even when protein substrate unfolding is not required such as in
peroxisomal protein translocation. In such cases additional roles for molecular
chaperones such as in targeting substrate proteins or regulating import machinery need to
be considered. The presence of peptide signals in substrate proteins and the removal of
these signal sequences after import is also a common, but not universal, finding. Such
targeting peptides exist for chaperone-mediated autophagy, for example, but removal of
such sequences is not necessary in a protein degradation pathway in which substrate
proteins do not have to function within the organelle.
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