Báo cáo khoa học: Trafficking and proteolytic processing of RNF13, a model PA-TM-RING family endosomal membrane ubiquitin ligase pot

MINIREVIEW
Trafficking and proteolytic processing of RNF13, a model
PA-TM-RING family endosomal membrane ubiquitin ligase
Jeffrey P. Bocock
1
, Stephanie Carmicle
2
, Mayukh Sircar
1
and Ann H. Erickson
1
1 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA
2 Department of Biological Sciences, Mississippi College, Clinton, MS, USA
Physiological function of RNF13
RING finger protein 13 (RNF13) has been linked to a
variety of physiological conditions through its isolation
in multiple screens for functional genes. The C-terminal
half of the protein contains a RING domain that func-
tions as an ubiquitin ligase in vitro [1,2]. Presumably,
the ability of RNF13 to ubiquitinate and so determine
the half-life and ⁄ or targeting of other proteins is
central to its physiological role, but substrates of this
ubiquitin ligase have not yet been identified. Expression
levels of the protein are higher in adult tissues than in
the corresponding embryonic tissues [2,3], suggesting
roles in development, consistent with the absence of
homologs in yeast. Expression of RNF13 is upregulat-
ed when mouse brain neurons are treated with tenas-
cin C [4], in basilar papilla when chickens are exposed
to acoustic trauma [5], in pancreatic and other tumors
[1,6], when neurons are stimulated to extend neurites

on fibronectin [2] and on treatment of chicken fetal
myoblasts with myostatin [3]. RNF13 has been
reported to promote neurite extension when over-
expressed ectopically in PC12 rat adrenal medulla
pheochromocytoma cells cultured on collagen [7].
Keywords
cellular targeting; endosomes; GRAIL; inner
nuclear membrane; protease-associated
domain; proteasomes; proteolysis; RING
domain; RMR; ubiquitin ligase
Correspondence
A. H. Erickson, The Department of
Biochemistry and Biophysics, CB 7260 GM,
University of North Carolina, Chapel Hill,
NC 27599, USA
Fax: +1 929 966 2852
Tel: +1 919 966 4694
E-mail:
(Received 17 April 2010, revised 2 July
2010, accepted 15 July 2010)
doi:10.1111/j.1742-4658.2010.07924.x
RING finger protein 13 (RNF13) is a ubiquitously expressed, highly regu-
lated ubiquitin ligase anchored in endosome membranes. A RING domain
located in the cytoplasmic half of this type 1 membrane protein mediates
ubiquitination in vitro but physiological substrates have not yet been identi-
fied. The protein localized in endosomal membranes undergoes extensive
proteolysis in a proteasome-dependent manner, but the mRNA level can
be increased and the encoded protein stabilized under specific physiological
conditions. The cytoplasmic half of RNF13 is released from the membrane
by regulatory proteases and therefore has the potential to mediate ubiquiti-

nation at distant sites independent of the full-length protein. In response to
protein kinase C activation, the full-length protein is stabilized and moves
to recycling endosomes and to the inner nuclear membrane, which exposes
the RING domain to the nucleoplasm. Thus RNF13 is a ubiquitin ligase
that can potentially mediate ubiquitination in endosomes, on the plasma
membrane, in the cytoplasm, in the nucleoplasm or on the inner nuclear
membrane, with the site(s) regulated by signaling events that modulate
protein targeting and proteolysis.
Abbreviations
APP, amyloid precursor protein; CTF, C-terminal fragment; EGFR, epidermal growth factor receptor; GRAIL, gene related to anergy in
lymphocytes; HA, hemagglutinin; ICD, intracellular C-terminal domain; INM, inner nuclear membrane; MVB, multivesicular body; NLS,
nuclear localization signal; PA, protease-associated; PKC, protein kinase C; PM, plasma membrane; PMA, 4b-phorbol 12-myristate
13-acetate; RMR, receptor homology region-transmembrane domain-RingH2 motif protein; RNF13, RING finger protein 13; TM,
transmembrane.
FEBS Journal 278 (2011) 69–77 ª 2010 The Authors Journal compilation ª 2010 FEBS 69
Overexpression of RNF13 suppresses cell proliferation
[3] but drives Matrigel invasion [1]. It remains unclear
how these seemingly disperate effects are mediated on
a biochemical level, but involvement of the extracellu-
lar matrix appears to be a recurring theme.
Cellular localization of RNF13
Substrate choice of a ubiquitin ligase is regulated by
its cellular localization. Using immunofluorescence
staining, we observe RNF13 at a steady state in
multivesicular endosomes, where it shows partial
colocalization with CD63, LAMP2 and the mannose
phosphate receptor [2]. Ectopically expressed protein
has also been detected in the endoplasmic reticulum
[1]. RNF13 is often observed in ring-shaped structures,
consistent with localization in the membrane of vesicles

(Fig. 1). Two PA-TM-RING family members, RNF128 ⁄
gene related to anergy in lymphocytes (GRAIL) [8,9]
and receptor homology region-transmembrane domain-
RingH2 motif protein (RMR) [10,11] are also localized
in endosomes.
Proteolytic processing of RNF13
RNF13 undergoes extensive post-translational proteol-
ysis (Fig. 2A). Unless expression levels are artificially
high because of transient ectopic expression, we are
generally unable to detect the expressed protein in cell
extracts by PAGE (Fig. 2B, lanes 1 and 4) or in intact
cells by immunofluorescence [2]. Inhibition of lyso-
somal proteolysis by modulation of pH does not pre-
vent generation of the proteolytic fragments or
significantly stabilize them [2]. Inhibiting the protea-
some does not prevent proteolysis of RNF13, but
unlike inhibition of lysosomal proteolysis, does retard
turnover of fragments. Thus the RNF13 fragments
must be generated by specific regulatory proteases and
are not merely by-products of protein turnover.
Inhibition of proteasome proteolysis by treatment of
cells with MG132 (Fig. 2, lanes 2 and 5) or epoxomi-
cin (Fig. 2, lanes 3 and 6) for  8 h allows sufficient
accumulation of protein to render RNF13 detectable
Fig. 1. RNF13 is localized in ring-shaped structures. Chinese ham-
ster ovary cells stably expressing FLAG-tagged RNF13 were
stained with mouse anti-FLAG IgG followed by donkey AlexaFluor
488 anti-mouse IgG (green) and goat anti-(lamin B) IgG followed by
donkey AlexaFluor 568 anti-goat serum (red). The inset shows an
enlargement of the ring-shaped vesicles.

Dimethylsulfoxide
MG132
Epoxomicin
Dimethylsulfoxide
MG132
1 2 3 4 5 6
72 kDa-
36 kDa-
55 kDa-
Full-Length
+ Chondroitin
sulfate
CTF-43
ICD
CTF-39
CTF-37
Epoxomicin
Anti-HA
Anti-FLAG
B
A
SP
PA
RING
PEST
TM
Ser-rich
CTF
ICD
Fig. 2. RNF13 undergoes extensive post-translational proteolysis.

(A) RNF13 is predicted to possess a transient N-terminal signal
peptide (SP), a protease-associated (PA) domain, a hydrophobic
transmembrane sequence (TM), and a RING, PEST and Ser-rich
domain. The membrane-bound C-terminal fragment(s) (CTF) and
the soluble intracellular C-terminal domain (ICD) are indicated. (B)
Chinese hamster ovary cells stably expressing RNF13-HAF were
treated for 10 h with vehicle (dimethylsulfoxide) (lanes 1 and 4) or
with a proteasome inhibitor, either 5 l
M MG132 (lanes 2 and 5) or
0.5 l
M epoxomicin (lanes 3 and 6). Cellular proteins were resolved
on a 12% NuPage Novex bis-Tris polyacrylamide gel (Invitrogen,
Carlsbad, CA, USA) run in Mes buffer and RNF13 was visualized on
a western blot with horseradish peroxidase-conjugated anti-HA or
anti-FLAG IgG, as indicated. Prestained molecular mass markers
are indicated on the left. The identity of the forms, as labeled on
the right, was established by blotting microsomal membranes with
antisera specific for the epitope tags [2].
RNF13 trafficking and proteolysis J. P. Bocock et al.
70 FEBS Journal 278 (2011) 69–77 ª 2010 The Authors Journal compilation ª 2010 FEBS
in cells that stably express the protein, suggesting a
significant role for proteasomes in the turnover of this
endosomal protein. MG132 has been used similarly to
stabilize and so aid visualization of biosynthetic forms
of other integral membrane proteins including Notch
[12], EGF receptor [13], growth hormone receptor [14]
and the amyloid precursor protein (APP) [15]. Epox-
omicin is a specific inhibitor of the chymotryptic-like
activity of the proteasome [16]. MG132, however, not
only blocks proteosome proteolysis, but also inhibits

calpain [17] and lysosomal cysteine proteases [18].
Thus MG132 potentially achieves its greater stabiliza-
tion of RNF13 relative to epoxomicin (Fig. 2, lane 5
versus lane 6) by inhibiting more than one protease
that cleaves the protein.
To determine the relationship of the biosynthetic
forms stabilized by proteasome inhibitors, we added
epitope tags to RNF13, marking the N-terminal ecto-
domain with a hemagglutinin (HA) epitope at position
38, after the signal peptide cleavage site predicted by
the algorithm signalp v.3.0 [19], and tagging the cyto-
plasmic half of the protein with a 3· FLAG epitope
inserted at the C-terminus after amino acid 381 [2].
The resulting protein was designated RNF13–HAF.
Antiserum specific for the HA epitope recognizes only
full-length RNF13 and full-length protein modified
with chondroitin sulfate (Fig. 2, lanes 2 and 3). Antise-
rum specific for the C-terminal FLAG epitope recog-
nizes full-length RNF13 as well as C-terminal
fragments (CTFs) and the intracellular C-terminal
domain (ICD) (Fig. 2, lanes 5 and 6).
Proteolytic cleavage of the N-terminal protease-asso-
ciated (PA) luminal domain or ectodomain must occur
because 43 and 39 kDa membrane-bound CTFs lack-
ing the HA tag are detected when microsomal mem-
branes isolated from B35 rat neuroblastoma cells
treated with MG132 are stripped of peripheral proteins
(Fig. 3, lanes 3 versus 5). A third CTF (37 kDa) is
detected if cells are treated with epoxomicin (Fig. 2,
lane 6). MG132 stabilizes CTF-39 efficiently, but this

fragment is barely detectable in epoxomicin-treated
cells; epoxomicin stabilizes CTF-37, but this form is
hard to detect in MG132-treated cells (Fig. 2, lanes 5
versus 6). The fact that the CTFs differ slightly in size
is consistent with the possibility that they are gener-
ated by different ectodomain-cleaving proteases.
Additional fragments of RNF13 are variably
detected, such as an HA-tagged form lacking the
C-terminal FLAG epitope (Fig. 3, lane 5, asterisk) [2],
suggesting that additional cleavages can occur under
specific conditions. The regulation of other PA-TM-
RING proteins by proteolysis has not been described,
although multiple forms of the homolog h-Goliath
have been detected by in vitro translation in the pres-
1 2 3 4 5
Sol Mb Cells Mb
Anti-HA
Anti-FLAG
54-
38-
*
ICD
Full-length
CTFs
Fig. 3. Proteolysis releases the cytoplasmic half of RNF13 from
the membrane. Microsomal membranes were prepared [2] from
MG132-treated B35 rat neuroblastoma cells [61] stably expressing
RNF13–HAF. Proteins in the soluble cytoplasmic fraction (lane 1,
Sol), in the soluble fraction generated when microsomal mem-
branes (Mb) were stripped of peripheral proteins (lane 2), in the

stripped microsomal membranes (lanes 3 and 5), and in whole cells
(lane 4) were resolved on a 12% Laemmli polyacrylamide gel. Bio-
synthetic forms of RNF13 were visualized on a western blot with
anti-(FLAG-horseradish peroxidase) or anti-(HA- horseradish peroxi-
dase) IgG, as indicated. Prestained molecular mass markers are
indicated on the left.
C
D
E
B
+PMA
RNF13
Lamin B
Merge
A
5 µm
5 µm
5 µm
5 µm
5 µm
+Dimethylsulfoxide
Fig. 4. Phorbol ester-stabilized RNF13 can target to the inner nuclear
membrane. Chinese hamster ovary cells (A,B) stably expressing
RNF13–HAF were treated with vehicle (dimethylsulfoxide) (A) or with
1 l
M 4b-phorbol 12-myristate 13-acetate (PMA) (B) for 6 h. RNF13–
HAF expression was detected with mouse anti-HA IgG followed by
donkey Alexa Fluor 568 anti-mouse serum. HeLa cells (C–E) tran-
siently expressing RNF13–HAF were serum-starved for 2 h and trea-
ted with 1 l

M PMA for 4 h. RNF13 expression was detected with
mouse anti-FLAG IgG followed by donkey AlexaFluor 568 anti-mouse
serum. The inner nuclear membrane protein lamin B was stained
with goat anti-(lamin B) IgG, followed by donkey AlexaFluor 488 anti-
goat serum.
J. P. Bocock et al. RNF13 trafficking and proteolysis
FEBS Journal 278 (2011) 69–77 ª 2010 The Authors Journal compilation ª 2010 FEBS 71
ence of microsomal membranes [20], suggesting that
proteolysis initiates in the endoplasmic reticulum or
cis-Golgi. The extensive post-translational proteolysis
of RNF13 occurs constitutively in cultured cells but is
potentially regulated by physiological conditions
in vivo. Proteolytic processing adds the potential for
temporal control of ubiquitination activity, but it
could also alter the cellular site of the ligase activity
and thus control the potential substrate population.
Post-translational modification of
RNF13
RNF13 in cell extracts migrates as a 60 kDa [1] to
65 kDa [2] protein on PAGE. Pulse–chase analysis
established that this is the initial biosynthetic form of
RNF13 [2], but this is larger than predicted by the
amino acid sequence, suggesting extensive post-trans-
lational modification occurs. As expected, one to two
asparagines in the luminal domain acquire high-
mannose carbohydrate that becomes modified with
complex sugars [1,2]. RNF13 is also modified by addi-
tion of heterogeneous chondroitin sulfate glycosamino-
glycan chains [2], producing a smear of protein bands
migrating above full-length protein (Fig. 2). Proteogly-

can is similarly added to integral membrane proteins
which localize to endosomes and the plasma membrane,
such as APP [21] and the immunoglobulin invariant
chain [22,23]. Because most ubiquitin ligases self-
ubiquitinate, it is also possible that some of the high-
mass forms detected when proteasomal proteolysis is
inhibited are heterogeneously ubiquitinated full-length
RNF13.
Expression in bacteria of a D1–205 RNF13 con-
struct, which lacks the ectodomain and the transmem-
brane (TM), generates a protein that migrates at
28 kDa, not at the expected 20 kDa, despite the
absence of post-translational modification in prokary-
otic cells [2]. When a similar construct is expressed in
eukaryotic cells, it comigrates with the 38 kDa ICD
[2]. Modifications that might contribute to the molecu-
lar mass of the C-terminal tail in eukaryotic cells, but
have not yet been identified on RNF13, include phos-
phorylation and tyrosine sulfation, observed on APP
[24], which is also a type 1 endosomal integral mem-
brane protein, ubiquitination of the PEST sequence [2]
or other lysines in the C-terminal half of the protein,
and methylation, acetylation and sumoylation.
PA domain cleavage
The fate of the PA domain released from the mem-
brane by proteolysis is unknown. If cleavage occurs on
the endosomal membrane, the domain could be rapidly
degraded in lysosomes. If cleavage occurs on the
plasma membrane or if vesicles containing cleaved
RNF13 fuse with the plasma membrane, the soluble

PA domain could be released outside the cell. Under
steady-state conditions, most of the protein resides
on endosomes so the percentage released to the cell
exterior is expected to be small, but physiological sig-
nals could change the amount of proteolysis or the
protein location relative to proteases.
The PA domain has been predicted to be a protein
interaction domain [25,26], but proteins which interact
with the PA domain, either as part of the full-length
RNF13 protein or as a solubilized domain, have not
yet been identified. In analogy to epidermal growth
factor receptor (EGFR) [27], the particular ligand
bound could ultimately determine the cellular targeting
and fate of the RNF13 molecules. RNF13 upregula-
tion initiates in response to extracellular signals such
as tenascin C and myostatin, but there is no evidence
the ubiquitin ligase directly binds these molecules.
In analogy to GRAIL [28], the RNF13 PA domain
may bind the lumenal domain of integral membrane
proteins which it subsequently ubiquitinates on the
cytoplasmic side of the membrane. In analogy to plant
RMR [11], RNF13 PA domain could bind proteases in
the Golgi and mediate their targeting to multivesicular
endosomes. Because ligands are commonly released by
the acidic pH of endosomal vesicles, these two roles
need not be mutually exclusive.
The shed RNF13 PA ectodomain could, in analogy
to EGFR superfamily members, serve extracellularly
as a ligand that either acts as a juxtacrine factor or
mediates transactivation of a distant unknown recep-

tor. Ectodomain cleavage would terminate RING-med-
iated ubiquitination if the PA domain selects targets.
Activity would also be terminated if the PA domain
mediates dimerization critical for function, this protein
domain does in tomato subtilase [29,30]. Alternatively,
it is possible that in vivo the cleaved PA domain
remains associated with a CTF, as observed for the
NOTCH heterodimer [31].
Liberation of the C-terminal tail
The C-terminal cytoplasmic domain of RNF13 is also
shed from the membrane and can be detected as a
soluble protein, the ICD, in the cytoplasmic fraction
when microsomal membranes are prepared from
MG132-treated cells (Fig. 3, lane 1). Full-length
RNF13, including protein modified with chondroitin
sulfate, and the CTF forms remain in membranes when
they are stripped of peripheral proteins (Fig. 3, lanes 3
RNF13 trafficking and proteolysis J. P. Bocock et al.
72 FEBS Journal 278 (2011) 69–77 ª 2010 The Authors Journal compilation ª 2010 FEBS
and 5), indicating that these are all integral membrane
proteins. Cleavage presumably occurs very near the
membrane surface because the ICD comigrates on
PAGE with the ectopically expressed cytoplasmic frag-
ment RNF13d1–206, generated by insertion of a Met
before Val207 [2]. The N-terminal residue of the
endogenous ICD is not known because the precise
cleavage site has not been determined. To escape rapid
degradation, a polypeptide must begin with Met or
Val, according to the N-end rule [32,33]. If the ICD
initiates with Met202, localized at the TM–cytoplasmic

junction, the ICD may evade N-terminal ubiquitination
[33] and thus be sufficiently stable to mediate function.
This proteolytic processing resembles that of ErbB4
[34]. For this EGFR family member, release from the
membrane by proteolysis constitutes a switch from
activation of one pathway by signaling as a TM
protein to initiation of new functions mediated by the
soluble ICD in a different cellular compartment [33].
Two different algorithms, PredictNLS [35] and
pSORT II [36], predict that the mouse RNF13 ICD
contains a nuclear localization signal (NLS) N-termi-
nal to the RING domain. PredictNLS designates the
sequence RRNRLRKD as an NLS and predicts the
protein binds DNA. pSORT II predicts the NLS is
PVHKFKK. These two sequences, separated by five
amino acids, might function alone or as part of a
tripartite NLS similar to that recently described for
EGFR family members [37]. The cytoplasmic tails of
proteins released from the membrane by regulated
intramembrane proteolysis frequently undergo NLS-
mediated import into the nucleus, where they modu-
late transcription [33,38]. Thus it is possible that the
RNF13 ICD that possesses the RING domain partic-
ipates in modulation of transcription, perhaps con-
trolling the half-life of transcription factors. RNF13
was initially postulated to regulate gene expression in
the nucleus based on detection in the sequence of a
leucine zipper, a motif that often mediates protein
dimerization, a putative NLS, and the presence of a
region rich in acidic amino acids following the RING

domain [4]. The presence of a PEST sequence charac-
teristic of rapidly turned over proteins is consistent
with a role in regulation of transcription. Ectopically
expressed RNF13d1–206 does not target to the
nucleus [2], however, but interaction with other pro-
teins or post-translational modification such as phos-
phorylation may be required for protein stabilization
and targeting to the nucleoplasm under specific physi-
ological conditions. For example, the ICD of APP
must associate with a histone acetyltransferase,
Tip60, in order to be transported to the nucleus
[39,40].
Targeting to the inner nuclear
membrane
Treatment of cells with phorbol esters such as 4b-phor-
bol 12-myristate 13-acetate (PMA) activates protein
kinase C (PKC), an enzyme that regulates cell prolifer-
ation, differentiation, angiogenesis and apoptosis
through the ability of its isoforms to initiate key sig-
naling cascades at the plasma membrane (PM) [41].
Upon stimulation, PKCa and PKCbII and plasma
membrane receptors, such as EGFR and the transfer-
rin receptor, move to the pericentrion, a PKC-depen-
dent subset of Rab11-positive recycling endosomes
concentrated around the microtubule-organizing cen-
ter ⁄ centrosome [42–45]. PMA similarly induces RNF13
to accumulate in perinuclear recycling endosomes
(Fig. 4B,C), where it colocalizes with the transferrin
receptor [46]. In HeLa cells, a spherical unstained area,
characteristic of the centrosome, is often detected when

cells are stimulated with PMA and stained for RNF13
(Fig. 4C). RNF13 could reach recycling endosomes via
the PM or could be transported directly to recycling
endosomes from the trans-Golgi network. There is
increasing evidence supporting a role for recycling
endosomes in biosynthetic pathways [47–49]. Surpris-
ingly, on PMA treatment of cells,  20% of the
RNF13 additionally moves to the inner nuclear mem-
brane (INM), where it colocalizes with lamin B
(Fig. 4E) [46]. Trafficking to recycling endosomes is
required for subsequent transport to the INM, as
expression of dominant-negative Rab11 blocks nuclear
localization of RNF13 [46]. Full-length RNF13
possessing both epitope tags and RNF13 CTFs are
found in purified INM fractions [46].
This signal-induced movement to the INM places
the RING domain in the nucleoplasm and the PA
domain between the two nuclear membranes. The PA
domain could bind substrates at this site or possibly a
protein bound to the PA domain earlier in the biosyn-
thetic pathway might be transported to the membrane
space as a PA domain ligand. This unusual targeting
pathway has only been described for two PM-localized
epidermal growth factor family members, heparin-
binding epidermal growth factor (HB-EGF) [50] and
proamphiregulin [51]. Both regulate transcriptional
activity following localization in the INM. However,
they possess short cytoplasmic tails of < 25 residues
that are not removed by proteolysis. Selective seques-
tration of receptors in the pericentrion is thought to

protect them from agonist-induced degradation.
Consistent with this, this altered cellular location of
RNF13 coincides with an increase in protein stability
[46]. These changes in cellular location prolong the
J. P. Bocock et al. RNF13 trafficking and proteolysis
FEBS Journal 278 (2011) 69–77 ª 2010 The Authors Journal compilation ª 2010 FEBS 73
ubiquitin ligase activity of RNF13 and expose the
ubiquitin ligase to different substrates.
Trafficking of a PA-TM-RING protein
The complex cellular trafficking and regulation of
RNF13 by proteolysis is diagrammed in Fig. 5.
Transport of the newly synthesized protein across the
endoplasmic reticulum membrane is mediated by the
transient N-terminal signal peptide. High-mannose
carbohydrate added co-translationally is modified with
complex sugars in the Golgi and proteoglycan chains
are added. On exiting the trans-Golgi network, RNF13
can enter a constitutive pathway (Fig. 5A) or a signal-
induced pathway (Fig. 5B). In analogy to APP [24],
RNF13 may reach multivesicular bodies (MVBs) fol-
lowing transport to the PM, followed by endocytosis
into endosomes. The presence of a dileucine motif in
the cytoplasmic half of the mouse protein (amino acids
307–312) suggests that the protein is capable of under-
going endocytosis. Alternatively, RNF13 could travel
directly to MVBs in analogy to the plant homolog
RMR, which serves as a targeting receptor delivering
enzymes to protein storage bodies [11,52]. Once local-
ized in an MVB, RNF13 could mediate ubiquitination
of substrates on the endosomal membrane, binding

substrates with the lumenal PA domain. In addition, it
is well-established that in response to specific stimuli,
such as calcium ionophores, MVBs can fuse with the
plasma membrane [53,54]. This could position RNF13
on the PM. Endocytosis of PM-localized RNF13
might expose RNF13 to endosome-localized proteases,
resulting in solubilization of essentially the entire
cytoplasmic tail. The ICD could be turned over by
proteasomes or possibly, as a result of post-transla-
tional modification and ⁄ or association with interacting
proteins, the NLS could mediate import into the nucle-
oplasm. Here RNF13 could potentially mediate ubiq-
uitination, perhaps utilizing the Ser-rich C-terminal
domain to bind soluble substrates in the absence of the
PA domain.
In response to extracellular signals that activate
PKC, RNF13 can enter a regulated trafficking path-
way that ultimately delivers the protein to the INM.
Our studies indicate that it is newly synthesized
RNF13, not protein stored in MVBs, which enters this
pathway [46]. Following PKC treatment, the majority
of RNF13 localizes to recycling endosomes. Both full-
length and RNF13 CTFs ultimately reach the INM
[46], where they colocalize with lamin B, a component
of the inner nuclear membrane.
Thus RNF13 can potentially ubiquitinate substrates
in organelles of the biosynthetic pathway, such as the
endoplasmic reticulum, Golgi, PM or endosomes. In
addition, RNF13 has the potential to ubiquitinate two
distinct sets of nuclear proteins. Full-length RNF13

positioned in the INM could capture integral mem-
brane protein substrates via its N-terminal PA domain.
ICD soluble in the cytoplasm could capture nucleo-
plasm substrates via its C-terminal Ser-rich domain.
The two pathways targeting RNF13 to the nucleus
presumably lead to ubiquitination of distinct sets of
substrates. Thus a single ubiquitin ligase may ubiquiti-
nate different substrates under different physiological
conditions that alter its cellular localization.
This complex regulation by cellular targeting and
proteolysis is unique for ubiquitin ligases, which are
commonly soluble proteins, but similar to that
described for such physiologically important proteins
as Notch [31], members of the EGFR superfamily of
tyrosine kinases [55,56] and APP [24,57]. The growing
appreciation of the role of both the nuclear membrane
[58,59] and endosomes [60] in the regulation of tran-
scription suggests PA-TM-RING ubiquitin ligases are
well-positioned to impact key regulatory events of the
cell.
C
N
2
Cleavage
EE
C
N
Golgi
C
N

PM
Endocytosis
Secretion
Nucleus
C
ONM
INM
C
C
N
MVB
C
N
C
N
C
N
Golgi
C
N
RE
C
N
EE
C
N
ONM
INM
Nucleus
AB

Constitutive pathway
Signal-induced pathway
PM
Fig. 5. RNF13 targeting pathways.
RNF13 trafficking and proteolysis J. P. Bocock et al.
74 FEBS Journal 278 (2011) 69–77 ª 2010 The Authors Journal compilation ª 2010 FEBS
Acknowledgements
This work was supported by research grants
MCB-0235680 and MCB-0938796 (to AHE) from the
National Science Foundation. Microscopy was
performed at the University of North Carolina in the
Microscopy Services Laboratory, Department of
Pathology and Laboratory Medicine, under the
direction of C. Robert Bagnell, Jr. Cell sorting was per-
formed at the UNC Flow Cytometry Facility that is
under the direction of L. Arnold.
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