Tài liệu Báo cáo khoa học: The PA-TM-RING protein RING finger protein 13 is an endosomal integral membrane E3 ubiquitin ligase whose RING finger domain is released to the cytoplasm by proteolysis ppt

The PA-TM-RING protein RING finger protein 13 is
an endosomal integral membrane E3 ubiquitin ligase
whose RING finger domain is released to the cytoplasm
by proteolysis
Jeffrey P. Bocock
1
, Stephanie Carmicle
1
, Saba Chhotani
1
, Michael R. Ruffolo
1
, Haitao Chu
2
and
Ann H. Erickson
1
1 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA
2 Department of Biostatistics, University of North Carolina, Chapel Hill, NC, USA
Proteins of the PA-TM-RING family have a protease-
associated (PA) domain and a RING finger domain
separated by a transmembrane (TM) domain. PA
domains are 120–210 amino acid sequences located in
the noncatalytic regions of diverse proteases [1,2]. They
are found in multiple members of MEROPS peptidase
Keywords
E3 ubiquitin ligase; neurite outgrowth;
protease-associated domain; proteolysis;
RNF13
Correspondence
A. Erickson, 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 1 November 2008, revised 23
December 2008, accepted 20 January 2009)
doi:10.1111/j.1742-4658.2009.06913.x
PA-TM-RING proteins have an N-terminal protease-associated domain, a
structure found in numerous proteases and implicated in protein binding,
and C-terminal RING finger and PEST domains. Homologous proteins
include GRAIL (gene related to anergy in leukocytes), which controls
T-cell anergy, and AtRMR1 (receptor homology region-transmembrane
domain-RING-H2 motif protein), a plant protein storage vacuole sorting
receptor. Another family member, chicken RING zinc finger (C-RZF), was
identified as being upregulated in embryonic chicken brain cells grown in
the presence of tenascin-C. Despite algorithm predictions that the cDNA
encodes a signal peptide and transmembrane domain, the protein was
found in the nucleus. We showed that RING finger protein 13 (RNF13),
the murine homolog of C-RZF, is a type I integral membrane protein
localized in the endosomal ⁄ lysosomal system. By quantitative real-time
RT-PCR analysis, we demonstrated that expression of RNF13 is increased
in adult relative to embryonic mouse tissues and is upregulated in B35 neu-
roblastoma cells stimulated to undergo neurite outgrowth. We found that
RNF13 is very labile, being subject to extensive proteolysis that releases
both the protein-associated domain and the RING domain from the mem-
brane. By analyzing microsomes, we showed that the ectodomain is shed
into the lumen of vesicles, whereas the C-terminal half, which possesses the
RING finger, is released to the cytoplasm. This C-terminal fragment of
RNF13 has the ability to mediate ubiquitination. Proteolytic release of

RNF13 from a membrane anchor thus provides unique spatial and tempo-
ral regulation that has not been previously described for an endosomal E3
ubiquitin ligase.
Abbreviations
APP, Alzheimer’s precursor protein; AtRMR1, Arabidopsis thaliana receptor homology region-transmembrane domain-RING-H2 motif protein;
CHO, Chinese hamster ovary; C-RZF, chicken RING zinc finger; CTF, cytoplasmic C-terminal fragment; EEA1, early endosomal antigen 1; ER,
endoplasmic reticulum; GRAIL, gene related to anergy in leukocytes; HA, hemagglutinin; HAF, hemagglutinin and 3· FLAG epitopes; HRP,
horseradish peroxidase; ICD, intracellular domain; LAMP2, lysosomal-associated membrane protein 2; MPR, mannose 6-phosphate receptor;
MVB, multivesicular body; NLS, nuclear localization signal; PA, protease-associated; PDI, protein disulfide isomerase; PNGase F, peptide:
N-glycosidase F; RNF13, RING finger protein 13; TM, transmembrane.
1860 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
families [3], including the transferrin receptor, a cata-
lytically inactive protease, prostate-specific membrane
antigen [4], the human Golgi ⁄ endosomal signal pepti-
dase peptidase-like proteins SPPL2a and SPPL2b [5],
and streptococcal C5a peptidase [6]. PA domains have
been proposed to serve as substrate or ligand recogni-
tion domains [1] or as protease regulatory regions [2],
yet they have been functionally characterized only in
plant proteins. The BP-80 receptor, which targets pro-
teases to the plant lytic vacuole through recognition of
the NPIR sorting determinant, contains a PA domain.
Binding of vacuolar proteases requires the PA domain
as well as other regions of the BP-80 luminal domain
[7].
RING finger proteins constitute a subfamily of the
proteins that possess a pattern of cysteine and histidine
residues that chelate zinc ions. The RING subfamily is
thought to function exclusively in protein–protein
interactions rather than protein–nucleic acid interac-

tions [8]. Many RING finger proteins are E3 ubiquitin
ligases [9]. The ubiquitination system functions in a
variety of cellular processes, including protein degra-
dation and protein trafficking.
PA-TM-RING proteins that combine these two
domains have been identified in plants, Xenopus,
Drosophila and mammals, but not in yeast. The Arabi-
dopsis thaliana PA-TM-RING receptor homology
region–transmembrane domain–RING-H2 motif pro-
tein (AtRMR1) was found to colocalize with a protein
storage vacuole membrane marker and was predicted to
be a receptor mediating targeting to the plant storage
vacuole [10]. This organelle is a multivesicular body
(MVB) containing segregated compartments of lytic and
storage activity [11,12]. AtRMR1 was subsequently
determined to be responsible for sorting the bean stor-
age protein phaseolin to the protein storage vacuole
[13] and was shown to bind to C-terminal vacuolar
sorting determinants on tobacco chitinase and barley
lectin [14], establishing that in plants the PA domain can
serve as a ligand-binding domain.
The best-characterized mammalian PA-TM-RING
family member is RNF128 ⁄ gene related to anergy in
lymphocytes (GRAIL). GRAIL was first identified in a
screen for genes upregulated in anergic CD4
+
T-cells,
which are unresponsive to antigen rechallenge [15]. It
was further characterized as an E3 ubiquitin ligase that
localizes to recycling endosomes, and was later con-

firmed to be necessary for induction of T-cell anergy
[16,17]. RING finger protein 13 (RNF13) was first
designated chicken RING zinc finger (C-RZF), a pro-
tein upregulated when chicken embryo brain cells were
treated with the extracellular matrix component tenas-
cin-C [18]. The protein was also upregulated in basilar
papilla when chickens were exposed to acoustic trauma
[19]. A truncated splice variant that lacks a complete
RING-H2 domain was additionally identified in mice
[19] but was not characterized. On the basis of immu-
nofluorescence microscopy and nuclear fractionation
experiments, Tranque et al. [18] reported that RNF13
is a nuclear protein, even though the tmpred algo-
rithm [20] predicts that it has a TM domain. A recent
study established that RNF13 is an E3 ubiquitin ligase
whose expression is increased in pancreatic ductal ade-
nocarcinoma tissues, suggesting that the protein may
participate in pancreatic cancer development [21].
We show that RNF13 is synthesized as an endoso-
mal integral membrane protein rather than a soluble
nuclear protein, consistent with other members of the
PA-TM-RING family. We demonstrate that RNF13
mRNA is upregulated following initiation of neurite
outgrowth, thus expanding on an array study that
found RNF13 expression to be sufficient to induce
neurite outgrowth [22]. We show that RNF13 is sub-
ject to unexpected proteolysis that releases both the
PA domain and the RING domain from the mem-
brane, providing a biochemical basis for understanding
the regulation of this family of multimodular endo-

somal membrane E3 ubiquitin ligases.
Results
Domain structure of RNF13
RNF13 contains a number of protein domains likely
to regulate its localization and function (Fig. 1). The
first 34 amino acid residues at the N-terminus are pre-
dicted by the algorithm signalp v.3.0 [23] to function
as a transient signal peptide, suggesting that the newly
synthesized polypeptide is translocated across the
endoplasmic reticulum (ER) membrane cotranslation-
ally. Residues 56–162 (numbering based on alignment
in Fig. S1) have a high degree of sequence identity to
PA domains. netnglyc 1.0 [24] predicts that this
domain contains two N-linked glycosylation sites, resi-
dues 43 and 88. Consistent with synthesis on the ER,
the program tmpred [20] predicts that residues 182–
203 comprise a 22-residue integral membrane sequence,
indicating that RNF13 might be a type I membrane
protein. predictnls [25] predicts that RNF13 has a
nuclear localization signal (NLS) (RRNRLRKD) at
residues 214–221, in the cytoplasmic half of the pro-
tein, near the membrane. psort [26] also predicts that
RNF13 has an NLS (PVHKFKK) but at residues
227–233, a site C-terminal to that identified by
predictnls. Residues 240–292 form a RING-H2
domain. Contiguous with the RING-H2 domain is a
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1861
24-residue sequence (residues 284–307) predicted, with
a high probability score of 14.33 (significant if > 5),

by the algorithm pestfind [27] to be a PEST domain.
PEST domains, defined as hydrophilic stretches of at
least 12 amino acids having a high concentration of
proline, glutamic acid, serine, and threonine, are pro-
tein domains that direct rapid degradation and thus
are usually found in proteins with a short half-life [28].
The remainder of the C-terminal region is rich in ser-
ine residues, similar to transcription factor activation
domains. Multiple phosphorylation sites are predicted
in the cytoplasmic half of the protein both by netph-
osk1.0 [29] and by group-based phosphorylation
scoring (GPS) 1.1 [30,31].
Sequence alignment of RNF13 with other
PA-TM-RING proteins
Three PA-TM-RING proteins, plant AtRMR1, mouse
GRAIL and mouse RNF13, exhibit little overall
sequence identity, as shown in the alignment in Fig. S1.
Only approximately 12% of the amino acids are
identical between the three proteins, as determined by
tcoffee alignment [32]. Most of the conserved residues
(gray boxes) lie within either the PA domain or the
RING-H2 domain.
RNF13 is an E3 ubiquitin ligase
RING finger sequences frequently mediate ubiquitin
ligase activity [9]; however, at least three distinct roles
have been described for RING domains [33]. We there-
fore investigated whether the RING domain in the
cytoplasmic half of RNF13 was capable of catalyzing
polyubiquitination. The cytosolic domain of
RNF13D1–205 comprising residues 206–381, and thus

the entire RING-H2 domain, was expressed in bacteria
with or without the point mutation C266A. This muta-
tion was designed to inactivate E3 ubiquitin ligase
activity of the RING-H2 domain, as does mutation of
the same conserved cysteine in the RING domain of
the E3 c-Cbl [34]. The expressed proteins, which con-
tained N-terminal 6· His epitope tags, were purified
on Ni
2+
–nitrilotriacetic acid affinity columns, eluted,
and resolved by SDS ⁄ PAGE. An antibody against 6·
His recognized two proteins in a western blot of each
eluate (Fig. S2A, lanes 1 and 2), establishing that both
bands contained the N-terminal epitope tag. The lower
band could result from early termination, but the two
discrete bands were reproducibly equally intense. Thus,
it is more likely that C-terminal cleavage of the protein
by a bacterial enzyme produces the lower band. The
size difference of 2 kDa indicates that only approxi-
mately 18 residues are missing from the C-terminus.
As the RING domain of RNF13 is composed of resi-
dues 240–292 out of 381, both protein bands should
contain an intact RING-H2 domain. The truncated
RNF13 proteins eluted from the affinity columns were
resolved on polyacrylamide gels that were stained with
Coomassie Blue R250 to assess purity (Fig. S2B).
Eluted protein was assayed for ubiquitin ligase activity
without further purification.
When RNF13D1–205 was added to an in vitro ubiq-
uitination reaction mixture including ubiquitin, puri-

fied commercial E1 enzyme, and a commercial E2
enzyme, either UbcH5a, UbcH5c, or UbcH6, it was
able to catalyze the formation of polyubiquitin chains,
as shown by the appearance of a high molecular mass
ladder of protein bands (Fig. S2C, lanes 1–3). As there
were only four proteins present in this in vitro assay,
and one of them was ubiquitin itself, these data sug-
gest that, like many E3 ubiquitin ligases, RNF13 can
ubiquitinate itself. All three E2s assayed interacted
with RNF13, but UbcH6 appeared to produce more
polyubiquitination (Fig. S2C, lane 3). As expected by
analogy with c-Cbl, purified RNF13D1–205 C266A
was unable to catalyze polyubiquitination when added
to a similar assay (Fig. S2C, lanes 4–6), as seen by the
PA
NLS
RING Ser-RichPESTTM
HA
3XFLAG
56
162
182
240
284
307
Fig. 1. RNF13 is a PA-TM-RING protein composed of several domains that might regulate other proteins. RNF13 is predicted by TMPRED [20]
to be a TM protein, with the hydrophobic TM domain falling in the middle of the amino acid sequence (residues 182–203). Additional major
domains include a predicted signal peptide (residues 1–34), a luminal PA domain (residues 56–162), and a cytoplasmic RING-H2 domain (resi-
dues 240–292). The protein is also predicted to have an NLS (residues 214–221 or 227–233), a PEST sequence (residues 284–307), and a
serine-rich region predicted to be phosphorylated (residues 309–381). We prepared expression constructs containing one or more of the

following epitope tags: an HA tag at position 38, a FLAG tag at position 377, or a 3· FLAG tag at position 381.
Proteolytic regulation of RNF13 J. P. Bocock et al.
1862 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
failure to produce the characteristic polyubiquitin
ladder. This indicates that catalysis of polyubiquitin
chains is specific to the RING-H2 domain of purified
RNF13, as a single point mutant of a conserved cyste-
ine can abrogate E3 ligase activity. Failure to catalyze
polyubiquitination was also seen when assay mixtures
were prepared that lacked any E2 enzyme (Fig. S2C,
lanes 7 and 8). These data show that the RING finger
of RNF13 requires active E2 enzyme to function as an
E3 ubiquitin ligase. As expected, when any of the
other essential components of the reaction, including
the E1 or E3 enzyme, ATP, or ubiquitin, was not
included in the reaction, polyubiquitination did not
occur (data not shown).
RNF13 is an endosomal protein
RNF13 is predicted to have a TM domain and signal
peptide, suggesting that it is an integral membrane
protein in the secretory pathway. C-RZF was localized
to the nucleus in chicken embryo heart cells [18], but
RNF13 was recently reported to be present in the ER
and Golgi when expressed transiently in MiaPaca-2
pancreatic cancer cells [21]. As no other PA-TM-
RING protein has been found in the nucleus or the
ER, we performed immunofluorescence experiments to
determine the subcellular localization of mouse
RNF13 (Figs 2 and 3).
AB

GHI
DF
C
E
Fig. 2. Endogenous, transiently expressed and stably expressed RNF13 all show punctate staining consistent with localization to endoso-
mal–lysosomal vesicles. (A, B) Primary cortical neurons prepared from embryonic day 14.5 mouse embryos were treated with MG132 for
12 h. Endogenous RNF13 was detected with antibodies directed against the 14 amino acid C-terminal peptide of mouse RNF13. Staining
was observed with the use of secondary donkey anti-rabbit Alexa Fluor 488 serum. The size bar in (B) represents 10 lm. (C) PC12 cells sta-
bly expressing RNF13 were treated with MG132 for 12 h. RNF13 expression was detected with mouse anti-FLAG serum and, as secondary
antibody, donkey anti-mouse Alexa Fluor 568 serum. (D–F) COS cells were transiently transfected with the RNF13 expression plasmid
pSG5X-RNF13 FLAG377. RNF13 (D) was detected with rabbit anti-FLAG serum and, as secondary antibody, anti-rabbit Texas Red serum.
Cells were counterstained with mouse antibodies raised against PDI (E) and donkey anti-rabbit Alexa Fluor 488 serum. These panels are
merged in (F). The size bar in (D–F) represents 20 lm. (G–I) HeLa cells stably expressing RNF13 were treated with MG132 for 12 h. RNF13
(G) was stained with mouse anti-FLAG and donkey anti-mouse Alexa Fluor 488 sera. Calnexin staining (H) was observed with rabbit anti-
calnexin and goat anti-rabbit Alexa Fluor 568 sera. These panels were merged in (I). The size bar in (G–I) represents 5 lm. RNF13 did not
colocalize with either of the two ER markers.
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1863
RNF13 observed in embryonic mouse cortical neu-
rons using an antiserum specific for the C-terminal 14
amino acids of RNF13 showed punctate, non-nuclear
staining characteristic of endosomes and lysosomes
(Fig. 2A,B). To facilitate detection of RNF13 by
immunofluorescence and to enable us to determine the
origin of the biosynthetic forms detected by western
blotting, we constructed vectors to express RNF13
with an N-terminal hemagglutinin (HA) epitope and a
C-terminal FLAG tag. Stably expressed, epitope-
tagged RNF13 exhibited punctate staining in PC12
cells, which are derived from a pheochyromocytoma of

the rat adrenal medulla and are frequently used as a
model for neuronal differentiation (Fig. 2C). The same
pattern was observed when epitope-tagged RNF13 was
expressed either transiently in COS cells (Fig. 2D–F)
or stably in HeLa cells (Fig. 2G–I). Thus, ectopic
expression from the vectors utilized in this study does
not appear to alter the localization of RNF13 relative
to the endogenous protein.
RNF13 was recently reported to be localized in the
ER, on the basis of transient expression in pancreatic
tumor cells [21]. In contrast, we found that the protein
is not present in the ER, as it failed to colocalize with
MPR
Golgin 97
K
FED
C
G
M
N
O
JL
HI
AB
RNF13 Mer
g
e
LAMP2
CD63
EEA1

Marker
Fig. 3. RNF13 is localized in MVBs and
endosomes. COS cells (A–L) or HeLa cells
(M–O) were transiently transfected with
RNF13-FLAG377, which was detected using
rabbit anti-FLAG sera (B, E, H, K, N). Cells
were costained with mouse anti-human
Golgin 97 (A) serum, mouse anti-human
LAMP2 serum (D), mouse anti-human CD63
serum (G), mouse anti-human MPR serum
(J) or mouse anti-human EEA1 serum (M).
Primary antibodies were visualized with the
secondary antibodies donkey anti-mouse
AlexaFluor 488 serum (A, D, G, J), goat
anti-rabbit Texas Red serum (B, E, H, K),
donkey anti-rabbit AlexaFluor 488 serum (N)
and goat anti-mouse AlexaFluor 568 serum
(M). RNF13 colocalized with LAMP2 (F),
CD63 (I), and MPR, (L), but not with Gol-
gin 97 (C) or EEA1 (O). Images were
obtained with a Zeiss LSM 210 confocal
microscope. The size bars represent 10 lm
(A–C, J–L) and 20 lm (D–I).
Proteolytic regulation of RNF13 J. P. Bocock et al.
1864 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
two different ER chaperone proteins. RNF13 did not
colocalize with endogenous protein disulfide isomerase
(PDI) when expressed transiently in COS cells
(Fig. 2D–F). Similarly, RNF13 expressed stably in
HeLa cells did not colocalize with calnexin (Fig. 2G–

I). Consistent with this, RNF13 did not accumulate
with the trans-Golgi network protein golgin 97
(Fig. 3A–C), indicating that our ectopically expressed,
epitope-tagged RNF13 is able to traverse the secretory
pathway efficiently.
Our immunofluorescence confocal microscopy
studies indicated that RNF13 is localized in the endo-
somal–lysosomal system (Fig. 3). RNF13 showed
significant colocalization with lysosomal-associated
membrane protein 2 (LAMP2), which localizes to the
membranes of endosomes and lysosomes (Fig. 3D–F).
RNF13 also partially colocalized with CD63
(Fig. 3G–I), a tetraspanin that localizes to multivesic-
ular endosomes [35], and with mannose 6-phosphate
receptors (MPRs) (Fig. 3J–L), which are enriched in
late endosomes. RNF13 failed to colocalize with the
early endosomal tether early endosomal antigen 1
(EEA1) (Fig. 3M–O) at several planes of depth in the
cell. Consistent with this, RNF13 did not colocalize
with fluorescently labeled transferrin internalized for
either 7.5 or 30 min by receptor-mediated endocytosis
(data not shown).
No accumulation of RNF13 in the nucleus could be
detected at steady-state by immunofluorescence stain-
ing of primary neurons or of cells expressing the pro-
tein either stably or transiently (Figs 2 and 3).
Similarly, nuclear RNF13 was not observed in pancre-
atic cancer cells transiently expressing RNF13 [21].
RNF13 undergoes extensive post-translational
proteolysis

To characterize the biosynthetic processing of RNF13,
we constructed viral expression vectors encoding
mouse RNF13 with an HA epitope at position 38 and
a3· FLAG epitope at position 381 (RNF13-HAF)
that we used to infect Chinese hamster ovary (CHO)
cells to produce the CHO-RNF13-HAF cell line, which
stably expresses RNF13. FLAG-positive RNF13-spe-
cific bands were not detected by western blot analysis
of cells expressing empty vector (Fig. 4A, lane 1). Sur-
prisingly, RNF13-specific FLAG-positive bands were
barely detectable in cell lysate when cells stably
expressing RNF13 were treated with dimethylsulfoxide
vehicle for 8 h (Fig. 4A, lane 2). When these cells were
incubated with the protease inhibitor MG132 in
dimethylsulfoxide for 8 h prior to harvest, however, a
specific RNF13 banding pattern indicative of extensive
post-translational modification was reproducibly
45” NS
1 2
1 2 3
-
-
-
1
2
3
97
37
54
kDa

Anti-FLAG
AB C
Anti-HA
1 2 3 4 5
RNF13
DMSO
MG132
RNF13 – + + + +
+ – + + +
+ – – +
–
–
–
+
+
–
+
+
+ +
– – – + +
DMSO
MG132
Epoxomicin
Anti-FLAG
Fig. 4. RNF13 undergoes extensive post-translational proteolysis. (A) CHO cells (lane 1) or CHO cells stably expressing RNF13-HAF (lanes 2
and 3) were treated, as indicated, with dimethylsulfoxide (DMSO) or MG132 for 8 h. Equal quantities of cellular protein were resolved on a
12% polyacrylamide gel. Biosynthetic forms of RNF13 were visualized on a western blot with mouse anti-FLAG serum. Prestained molecular
mass markers are indicated on the left. (B) CHO cells (lane 1) or CHO cells stably expressing RNF13-HAF (lanes 2–5) were treated, as indi-
cated, with dimethylsulfoxide, MG132 or epoxomicin for 10 h. RNF13 was visualized with anti-HA serum. (C) CHO cells transiently express-
ing pSG5X-RNF13-HAF were pulse-labeled with [

35
S]methionine for 45 min. RNF13 was immunoprecipitated with anti-FLAG serum and
resolved on a 12% polyacrylamide gel (lane 1). To detect nonspecific protein bands, normal whole serum (NS) was substituted for specific
affinity-purified anti-FLAG serum (lane 2).
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1865
detected (Fig. 4A, lane 3). The pattern included a het-
erogeneous collection of proteins of approximately
80 kDa, a second series of proteins that occasionally
resolved into four discrete bands at approximately
65 kDa (e.g. Fig. 5A, lane 2), three protein bands of
approximately 45 kDa, and one protein band of
approximately 36 kDa. As all these proteins were visu-
alized with antiserum that recognizes the 3· FLAG
epitope at residue 381, they all contain the C-terminus
of RNF13. An identical protein pattern was obtained
when RNF13-HAF was expressed stably in B35 rat
neurons (data not shown).
We next utilized antiserum specific for the N-termi-
nal HA epitope tag (residue 38) to determine which of
the RNF13 proteins in the banding pattern contain the
N-terminus. Cells were treated as indicated (Fig. 4B).
The specific proteasome inhibitor epoxomicin stabi-
lized RNF13 (Fig. 4B, lane 5), as did MG132 (Fig. 4B,
lane 4). Both the heterogeneous bands at  80 kDa
and the group of bands at  65 kDa were recognized
by the anti-HA serum (Fig. 4B, lanes 4 and 5). As
these proteins are also recognized by the anti-FLAG
serum, they must possess both residues 38 and 381 and
therefore be close to full-length RNF13. The lower

molecular mass bands around 45 kDa and at 36 kDa
were not recognized with anti-HA serum, suggesting
that the N-terminal portion of the protein containing
the HA epitope was lost from these proteins by pro-
teolysis.
To determine which of the RNF13 bands is the ini-
tial biosynthetic product, we pulsed transiently trans-
fected CHO cells expressing RNF13-HAF with
[
35
S]methionine and immunoprecipitated RNF13 using
antibodies specific for the FLAG epitope (Fig. 4C).
The major protein detected after a 45 min pulse
migrated at 65 kDa (Fig. 4C, lane 1). This protein
band was absent upon immunoprecipitation with nor-
mal serum as a negative control (Fig. 4C, lane 2).
RNF13 acquires carbohydrate modification
As we observed forms of RNF13 that migrated more
slowly on polyacrylamide gels than the 43 kDa form
predicted by the primary sequence alone, we assayed
the protein for sugar modification. The netnglyc 1.0
algorithm predicts that RNF13 possesses two
sequences in the N-terminal domain that could acquire
N-linked carbohydrate. Transiently expressed RNF13
was immunoprecipitated and treated with peptide:
N-glycosidase F (PNGase F), which removes both
asparagine-linked high-mannose and complex oligosac-
charides [36]. The 65 kDa region resolved, on this gel,
into four distinct proteins in the absence of endogly-
cosidase treatment (Fig. 5A, lane 2). After endoglycosi-

dase treatment, the two upper protein bands
disappeared, with a concomitant increase of the lowest
band. Identical results were obtained with drug prepa-
rations from two different suppliers (Fig. 5A, lanes 3
and 4). To confirm this result, CHO cells transiently
expressing FLAG-tagged RNF13 were cultured in the
presence of tunicamycin, an antibiotic that inhibits
transfer of N-acetylglucosamine 1-phosphate to doli-
cholmonophosphate [37], thus blocking the synthesis
of asparagine-linked oligosaccharide chains on glyco-
proteins. Tunicamycin treatment reproducibly reduced
the amount of the upper band and resulted in loss of
the middle band. These results confirm that two
N-linked sugar chains can be removed from RNF13,
supporting the predictions made by netnglyc 1.0.
As a percentage of certain integral membrane pro-
teins, such as the Alzheimer’s precursor protein (APP)
[38] and the immunoglobulin invariant chain [39,40],
acquire chondroitin sulfate glycosaminoglycan chains,
we also assayed RNF13 for this modification. RNF13
possesses one potential Ser-Gly dipeptide acceptor
sequence [41] in its luminal domain. When immuno-
precipitated RNF13 was treated with chondroitin-
ase ABC, the intensity of the diffusely staining bands
1 2 3 4 A
B
5
–+
Chondroitinase
Transfection +

Endo F –
T
unicamycin +
+
+
–
+
+
–
+
–
–
–
–
–
65 kDa
65 kDa
~80 kDa
12
Fig. 5. RNF13 is modified with N-linked sugars and chondroitin
sulfate. (A) pSG5X-RNF13-FLAG377 was expressed transiently in
CHO cells. Immunoprecipitated RNF13 was treated with PNGase F
from two different manufacturers (lanes 3 and 4). Transfected cells
were incubated overnight with tunicamycin to block high-mannose
sugar addition (lane 5). Cellular proteins were resolved on a 12%
gel, and RNF13 was identified by western blotting using an antise-
rum specific for the FLAG epitope. (B) Mouse RNF13-HAF was
expressed transiently in CHO cells, and immunoprecipitated with
antiserum specific for the FLAG epitope. The immunoprecipitate
was split into two equal parts, which were incubated overnight in

the absence (lane 1) or presence (lane 2) of chondroitinase ABC
prior to resolution on a 12% polyacrylamide gel.
Proteolytic regulation of RNF13 J. P. Bocock et al.
1866 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
at 80 kDa dramatically decreased, whereas the 65–
70 kDa bands increased in intensity (Fig. 5B). This
result indicates that at least a proportion of the
RNF13 protein is modified with chondroitin sulfate.
Proteolysis releases N-terminal and C-terminal
fragments of RNF13 from the membrane
To further characterize the 36 kDa FLAG-positive
RNF13 band observed in cell lysates, CHO cells were
transfected transiently with a construct that encodes
only the C-terminal half of RNF13. This variant
(RNF13D1–204) is initiated a few residues beyond the
putative TM sequence and retains the FLAG epitope.
It was found to comigrate with the 36 kDa protein in
cell lysates (Fig. 6A, lane 2 versus lane 4), suggesting
that the 36 kDa band is derived from full-length
RNF13 by proteolysis at or near the TM sequence. An
HA-positive protein of approximately the same size
was reproducibly detected when blots probed with
anti-HA serum were overexposed (Fig. 6B, lane 5).
This protein band always appeared fuzzy, consistent
with the presence of carbohydrate. Detection of this
protein suggests that the N-terminal domain of
RNF13, like the C-terminal domain, is released by
proteolysis from the TM anchor localized approxi-
mately in the middle of the protein.
RNF13 is a type I integral membrane protein

By isolating microsomes and stripping them of periph-
eral proteins, we confirmed the prediction of Mahon &
Bateman [1] that RNF13 is synthesized as an integral
membrane, not a nuclear, protein. We prepared micro-
somal membranes, by Dounce homogenization in the
presence of sucrose to maintain microsome integrity,
from a postnuclear supernatant of MG132-treated
CHO-RNF13-HAF cells. Proteins in both the postnu-
clear supernatant, which contains soluble cytoplasmic
proteins, and in the pelleted microsomes were resolved
on a polyacrylamide gel (Fig. 7A). A western blot was
probed for both the FLAG and HA epitopes. The
36 kDa protein, which comigrated with the expressed
soluble C-terminal cytoplasmic half of the protein
(Fig. 6A), was detected in the postnuclear superna-
tant ⁄ cytoplasmic fraction (Fig. 7A, lane 1), establish-
ing that the FLAG-tagged C-terminal half of RNF13
is released from the membrane by proteolysis and thus
resembles the intracellular domain (ICD) of other inte-
gral membrane proteins such as APP and Notch.
Recovery of the FLAG-tagged C-terminal fragment in
the cytoplasmic fraction also indicates that RNF13 is
a type I membrane protein that has its PA domain
either in the lumen of vesicles or on the cell surface
and its C-terminal half in the cell cytoplasm. All other
biosynthetic forms of RNF13, including the N-termi-
nal HA-tagged domain (Fig. 7B, lane 3), were present
in the microsome fraction, indicating they are either
embedded in the microsomal membrane or present
inside vesicles.

To confirm that RNF13 is an integral, not a periph-
eral membrane protein, we isolated microsomes from
CHO-RNF13-HAF cells and lysed them in high-pH
carbonate buffer (Fig. 7B). Freezing and thawing
microsomes in pH 11.5 buffer lyses vesicles and solu-
bilizes peripheral membrane proteins not embedded in
the membrane bilayer [42,43]. The luminal lysosomal
protease cathepsin L, detected as a control, was
present in the soluble fraction, confirming that soluble
content proteins are released by carbonate treatment
(data not shown).
All forms of RNF13 present in microsomes and
recognized by the anti-FLAG serum were present in the
membrane fraction and were not solubilized when vesi-
cles were lysed at high pH, indicating they are integral,
not peripheral, membrane proteins (Fig. 7B, lane 2).
The HA-tagged 36 kDa fragment of RNF13 was also
detectable within microsomes (Fig. 7B, lane 3), sug-
gesting that the luminal domain is shed within endo-
somes. With this protocol, an additional HA-positive
protein in the RNF13 pattern was reproducibly detect-
Ctl ICD RNF13
RNF13
12 3 4
5
Anti-FLA
ABnti-HA
-
-
GA

Virus +DMSO+MG132
+MG132
54
38
Fig. 6. RNF13 undergoes proteolysis on both sides of its trans-
membrane domain. (A) B35-Con cells (lane 1), B35-RNF13-HAF
cells (lanes 3 and 4) or CHO cells transiently expressing RNF13D1–
204, the ICD (lane 2), were treated, as indicated, with dimethylsulf-
oxide (DMSO) or with MG132 for 8 h. Equal quantities of protein
(600 lg) were loaded in lanes 1, 3 and 4, and 100 lg of protein
was loaded in lane 2. Biosynthetic forms of RNF13 were visualized
on a western blot of a 10% polyacrylamide gel with anti-FLAG-HRP
serum. (B) CHO-RNF13-HAF cells were treated with MG132 for
9 h, and RNF13 was visualized on a blot of a 12% gel with anti-HA
serum.
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1867
able in the membrane fraction below 65 kDa
(Fig. 7A,B, arrow). This protein, which lacks a
FLAG-tag and thus presumably has lost its C-terminal
sequences, was readily detectable in intact microsomes
(Fig. 7A), but was less apparent once microsomes were
lysed (Fig. 7B). It can often be visualized in whole cell
extracts after long exposure of the western blot to film,
suggesting that it corresponds to an authentic biosyn-
thetic form of RNF13 that is stable in intact micro-
somes (data not shown).
A model summarizing the major biosynthetic forms
of RNF13 and their relationship to membranes, based
on the observed molecular mass and presence or

absence of the epitope tags, is presented in Fig. 7C.
The three cytoplasmic C-terminal fragments (CTFs) or
‘stubs’ remaining after loss of the PA domain could be
generated by multiple proteases or could result from
multiple cleavages by one enzyme. Other biosynthetic
intermediates may be present but not detectable by our
gel system. Additionally, the ratio of the forms may
vary with the cell type and the metabolic condition of
the cells expressing RNF13.
Inhibiting the vacuolar ATPase only partially
stabilizes RNF13
Since RNF13 localizes to the endosomal–lysosomal
membrane system, we treated cells with two inhibitors
that raise the pH of vesicles in an attempt to inhibit
lysosomal proteolysis of RNF13. Bafilomycin A1
inhibits the vacuolar ATPase [44,45], and ammonium
chloride raises the pH of lysosomes and blocks the
light–heavy chain cleavage of lysosomal cathepsin L
[46]. Although MG132 is commonly employed as a
proteasome inhibitor, it has also been reported to
inhibit lysosomal cathepsins [47,48], calpains [47],
and BACE1 [49]. Stably expressed RNF13 was barely
detectable in cell extracts unless the cells were pretreated
with MG132 (Fig. 8A, lane 1 versus lane 2). Inhibition
of the vacuolar ATPase with bafilomycin A1 or by
treating cells with ammonium chloride (Fig. 8A, lanes
3 and 4, respectively) stabilized biosynthetic forms of
RNF13, but not as efficiently as did MG132 treatment
of cells. The data suggest that other proteases primar-
ily mediate the turnover of RNF13 in vesicles distinct

from mature lysosomes.
Ectopically expressed RNF13 ICD does not
localize in the nucleus when expressed
transiently from a plasmid
RNF13 is predicted to have one or two NLSs (Fig. 1),
but we detected only RNF13 in punctate structures by
confocal microscopy (Figs 2 and 3). Similarly, we were
unable to detect the FLAG-tagged 36 kDa ICD in
preparations of purified nuclei (data not shown). We
Anti-FLAG
1 2
1 2
Anti-HA
Cyto Mb
Cyto Mb
Cell fractionation
AB C
Lysed microsomes stripped of
peripheral proteins
Sol Mb Sol Mb Microsomes
1 2 1 2
Anti-FLAG
Anti-HA
Model for RNF13 Biosynthetic Forms
80 39-46 36
HA
HA
FLAG
HA
FLA

G
FLAG
FLA
G
70
FLAG
FLA
G
Full-length CTF ICD
~kDa:
63
97-
54-
37-
-
-
-
97
54
37
36
3
-
-
-
-
95
72
55
36

HA
Fig. 7. Proteolysis releases a C-terminal fragment of RNF13 into the cytoplasm. Microsomes were prepared by Dounce homogenization of
CHO cells stably expressing RNF13-HAF treated with MG132. (A) RNF13 in the soluble cytoplasmic fraction (lane 1, Cyto) and in the pelleted
microsome fraction (lane 2, Mb) was visualized by probing a blot with antibodies specific for the C-terminal FLAG or N-terminal HA epitope,
as indicated. (B) Pelleted microsomes were lysed and stripped of peripheral membrane proteins by resuspension and incubation in pH 11.5
carbonate buffer. RNF13 was visualized in the soluble (Sol) and membrane (Mb) fractions by probing a blot of a minigel with antibodies
specific for the C-terminal FLAG or N-terminal HA epitope, as indicated. The arrows mark an HA-tagged form that markedly decreases in
intensity when microsomes are lysed (B). This protein is present, but more difficult to resolve, on commercial minigels [(B), all lanes except
lane 3]. For the minigel, 100 lg of protein was resolved in each lane. (C) A model of the epitope-tagged protein bands detected is presented.
The three short horizontal lines on the full-length protein represent chrondroitin sulfate modification.
Proteolytic regulation of RNF13 J. P. Bocock et al.
1868 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
therefore transiently expressed RNF13D1–204 3·
FLAG381 (Fig. 9), containing only the C-terminal half
of RNF13 including the putative NLS, to determine
whether RNF13 could be observed in the nucleus
when expression of the ICD was high. Figure 9A,
showing cells treated with dimethylsulfoxide alone,
establishes the specificity of the anti-FLAG serum.
Despite the presence of two sequences predicted to be
NLSs, RNF13D1–204 3· FLAG381 localized to the
cytoplasm (Fig. 9C). This is in agreement with our cell
fractionation data, which also indicated that it is local-
ized to the cytosol (data not shown).
RNF13 expression is higher in adult than in
embryonic tissues
Genome sequencing suggests that RNF13 is ubiqui-
tously expressed. This is supported by expression data
from the Stanford Microarray Database, which show
RNF13 to be widely expressed in many cell types,

including throughout tissues of the human immune
and nervous systems [50]. However, initial northern
blot analysis of expression of C-RZF, the chicken
homolog of RNF13, showed that the protein was
expressed in embryonic heart and brain, but not in
liver [18]. We therefore analyzed mouse RNF13
expression by quantitative real-time RT-PCR, isolating
mRNA from both embryonic and adult mouse tissues.
The oligonucleotides used in this assay were specifi-
cally designed to bind only the full-length RNF13
transcript. Expression of RNF13 in adult heart tissue
was similar to that in spleen. We observed fold
increases of 5.7, 2.6 and 1.9 for adult kidney, liver and
brain, respectively, relative to spleen (Table 1; see
Table S3 for statistical analysis). The PA-TM-RING
family member GRAIL has been found to have similar
expression in mouse tissues, using northern blots [15],
but it has been primarily studied in T-cells. We also
observed that RNF13 expression levels in adult tissues
were higher than in the corresponding embryonic
tissue. For example, there was a four-fold increase in
adult brain as compared to embryonic brain after 14.5
or 16.5 days of development (Table 1). Our analysis of
embryonic tissue showed similar expression of RNF13
1 2 3 4
+MG132
+Bafilomycin
+DMSO
+NH
4

Cl
Anti-
α Tubulin
Anti-
A
B
FLAG
97
54
Fig. 8. Inhibitors stabilize RNF13 cleavage fragments. (A) CHO
cells stably expressing RNF13-HAF were treated, as indicated, with
dimethylsulfoxide (DMSO) (lane 1), MG132 (lane 2) or bafilo-
mycin A1 (lane 3) for 8 h, or with NH
4
Cl for 24 h (lane 4). Biosyn-
thetic forms of RNF13 were visualized on a western blot of a 12%
polyacrylamide gel with mouse anti-FLAG serum. Migration of pre-
stained molecular mass markers is indicated on the right. All lanes
shown are derived from the same exposure of the same blot. (B)
Equal quantities of total cellular protein were loaded in each lane,
as verified by blotting for a-tubulin.
RNF13 +DMSO RNF13 +MG132
RNF13 ICD
ABC
Fig. 9. Expressed RNF13 ICD is not localized in the nucleus. (A, B) CHO cells stably expressing RNF13-HAF were plated on coverslips, incu-
bated for 10 h with either dimethylsulfoxide (DMSO) vehicle (A) or MG132 (B), and stained with anti-FLAG serum, as indicated. (C) CHO
cells were transiently transfected with a plasmid encoding RNF13D1–204 3· FLAG381, the soluble ICD of RNF13, and stained with
anti-FLAG serum. RNF13 was visualized by confocal microscopy. The size bar in (C) represents 10 lm.
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1869

in both liver and brain, consistent with our findings in
adult tissue.
RNF13 is upregulated during neurite outgrowth
Quantitative real-time RT-PCR data indicated that
RNF13 is expressed in brain tissue (Table 1). A previ-
ous array study identified the RNF13 gene as one of
five genes that were able to promote neurite extension
when expressed ectopically in PC12 neuronal precursor
cells cultured on collagen [22]. The level of RNF13
expression was not assayed under these conditions. We
therefore determined whether endogenous RNF13 gene
expression increases during neurite outgrowth. B35
neuroblastoma cells were treated with dibutyryl-cAMP,
a reagent that stimulates neurite extension by these
cells [51], and RNF13 expression was analyzed using
quantitative real-time RT-PCR (Fig. 10). We observed
a two-fold increase in RNF13 mRNA after 72 h of
outgrowth. A similar two-fold increase of RNF13
expression was observed after 5 days of outgrowth.
Thus, RNF13 ubiquitin ligase activity may play a role
in the regulation of nerve cell development.
Discussion
We have determined that endosomal membranes pos-
sess an E3 ubiquitin ligase that can be released into
the cytoplasm by proteolysis. The PA-TM-RING pro-
tein RNF13 is synthesized as an integral endosomal
membrane protein, but post-translational proteolysis
solubilizes the C-terminal half of this type I membrane
protein. The role of ubiquitin addition in receptor
endocytosis and in the formation of multivesicular

endosomes is well established [52], but few endosomal
E3s have been characterized, and none has been shown
to be regulated by domain-specific proteolysis.
PA-TM-RING family members are predicted by
algorithms to be synthesized with transient signal
peptides, placing the proteins in the membranes of
the secretory pathway. RNF13 was recently reported
to reside in the ER and Golgi, on the basis of
immunofluorescence staining of cells transiently
expressing the protein [21]. In contrast, we found no
colocalization of mouse RNF13 with either of two
ER chaperones or with golgin 97, whether RNF13 is
expressed transiently or stably. Consistent with our
results, AtRMR1 and GRAIL are both present in
endosomes under steady-state conditions. The plant
family member AtRMR1 localizes to protein storage
vacuoles [10] and mouse GRAIL to recycling endo-
somes [15], although rat Goliath has been reported
to be present in mitochondria [53]. Because, unlike
GRAIL, RNF13 was not found in early endosomes,
as determined by the lack of costaining with the
early endosome tether EEA1, the two mouse proteins
have overlapping but different steady-state locali-
zations.
Proteolysis regulates the half-life of RNF13. Stably
expressed protein is barely detectable unless cells are
treated with protease inhibitors such as MG132 or the
Table 1. RNF13 expression in adult and embryonic tissues. Organs
were harvested from adult mice and from embryonic mice at
embryonic day (E) 14.5 and E16.5. RNF13 expression was normal-

ized using 18S rRNA as an internal control. Each RNA sample was
analyzed in duplicate, using quantitative real-time RT-PCR. Stati-
stical analysis of the data is presented in Table S3.
Group Organ
Mean
DCT
Fold
expression
Adult Brain 9.4 2.9
Adult Heart 10.3 1.5
Adult Kidney 8.1 6.7
Adult Liver 9.1 3.6
Adult Spleen 10.9 1.0
Adult Liver 9.1 11.5
E14.5 Liver 12.6 1.0
E16.5 Liver 11.2 2.7
Adult Brain 9.4 5.5
E14.5 Brain 11.8 1.0
E16.5 Brain 11.3 1.5
Fig. 10. RNF13 expression is increased following induction of neu-
rite outgrowth. B35 cells were plated on dishes coated with fibro-
nectin (5 mgÆmL
)1
) and incubated with dibutyryl-cAMP (100 lM)to
induce neurite outgrowth. RNA was harvested using Trizol reagent,
each plate being harvested separately as a single sample. Sample
numbers of n = 9 were collected for control cells, and sample num-
bers of n = 6 were collected for both time points of outgrowth.
Quantitative real-time RT-PCR was performed in duplicate for each
RNA sample, using ABI PRISM 7700 hardware and software. Data

were analyzed using the 2
DDCT
method. The error bars represent
the 95% confidence level. Statistical analysis is summarized in
Table S3.
Proteolytic regulation of RNF13 J. P. Bocock et al.
1870 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
proteasome inhibitor epoxomicin [54] prior to harvest.
MG132 has been used similarly to stabilize and so aid
visualization of biosynthetic forms of other integral
membrane proteins, including Notch [55], epidermal
growth factor receptor [55], growth hormone receptor
[56], and APP [49]. The regulation of other PA-TM-
RING proteins by proteolysis has not been described,
although multiple forms of h-Goliath have been
detected by in vitro translation [57].
By expressing RNF13 that was engineered with dis-
tinct epitope tags at its N-terminus and C-terminus, we
were able to determine the origin of its proteolytic
fragments. Our detection of the C-terminal domain
within the cytoplasm suggests that RNF13 may
undergo regulated intramembrane proteolysis, like
APP and Notch [58]. The endogenously generated
C-terminal fragment comigrated on polyacrylamide
gels with the ectopically expressed C-terminal half of
RNF13 (residues 206–381), indicating that the former
is essentially the entire cytoplasmic tail of RNF13.
Thus, cleavage must occur within or very near the TM
sequence. We have found that this ICD fragment can
mediate E3 ubiquitin addition in vitro, as can pancre-

atic RNF13 [21]. Interestingly, a splice variant of
human RNF13 (NM183383) has been identified that is
equivalent to this portion of RNF13.
Ectodomain shedding must precede proteolytic
cleavage within the membrane. The soluble HA-tagged
RNF13 fragment detected within microsomes, which is
approximately the size of the PA domain, is probably
primarily degraded in lysosomes, but it could be
secreted if the endosomes fuse with the plasma mem-
brane. A gene product equivalent to the PA domain
has been identified in mammalian cells (hPAP21), but
its physiological function is unknown [59]. The locali-
zation of RNF13 to MVBs or late endosomes sug-
gested that its N-terminal proteolysis might be
mediated by lysosomal enzymes. However, isoforms of
RNF13 were only slightly stabilized by the addition of
reagents that inhibit lysosomal proteases. Thus, other
cellular proteases must be primarily responsible for
mediating the turnover of RNF13.
RING E3s are commonly cytosolic, nuclear or
peripheral membrane proteins. The initial study of
RNF13 [18] concluded that the protein is localized in
the nucleus, on the basis of both immunofluorescent
staining and cell fractionation. The validity of the
fractionation protocol utilized has been questioned [1],
however, and the tmpred algorithm predicts that
RNF13 has a TM sequence. Consistent with this, our
analysis of cellular membranes washed with high-salt
solution establishes that RNF13 is indeed an integral
membrane protein. This is consistent with data showing

that RNF13 is enriched in the Triton X-114 phase when
cellular membranes are extracted with this nonionic
detergent [21]. Incorporation of the RING domain into
an integral membrane protein allows the E3 activity to
be spatially regulated. Presumably, the TM anchor
enables the enzymatic RING domain to be targeted to
the same cellular site as its substrate(s). Proteolytic pro-
cessing adds a potential for temporal control of enzy-
matic activity, but it could also alter the cellular site of
the E3 activity. The E3 enzyme could target endosomal
proteins or a substrate distant from the membrane of
the endosome. Thus, solubilized RING domain could
potentially exert functions independent of the full-
length, endosomally localized protein.
There is increasing precedent for proteolysis as a
means of mobilizing dormant transcription factors
[60]. On the basis of their detection of RNF13 in
chicken brain cell nuclei, Tranque et al. [18] suggested
that RNF13 might modulate transcription in embry-
onic chicken brain. The solubilization of the ICD of
RNF13 and the presence of the putative NLS on the
C-terminal side of the TM domain are consistent with
the possibility that the ICD, released from the mem-
brane by proteolysis, has a function in the cell nucleus.
We have, however, been unable to date to detect the
RNF13 ICD in the nucleus. There are at least three pos-
sible explanations for the absence of detectable RNF13
staining in the nucleus: (a) the PEST domain adjacent to
the RING domain is likely to ensure that the cytoplas-
mic tail, and thus the RNF13 E3 activity, has a short

half-life that is tightly regulated by ubiquitination and
proteasome degradation; (b) RNF13 may need a specific
signal to move into the nucleus either on its own or with
the help of an adaptor protein that facilitates entry into
or retention in the nucleus – it is possible that the adap-
tor protein is more abundant in certain cell types, or is
upregulated in response to an external stimulus, and
thus RNF13 cannot effectively move into the nucleus in
cultured cells; and (c) alternatively, RNF13 may not go
to the nucleus and instead may function as an E3 ligase
in the cytoplasm. Precedents exist for all three possibili-
ties. The ICDs of proteins such as Notch and APP are
notoriously difficult to detect in nuclei, as they represent
a small percentage of the whole protein and they are
very labile, so it remains possible that we are simply
unable to detect the small amount of ICD that might
localize to the nucleus constitutively. The APP ICD
must form a complex with the nuclear adaptor Fe65 and
the histone acetyltransferase Tip60 in order to target to
the nucleus [61,62]. Other ICDs function exclusively in
the cytoplasm. The adhesion protein N-cadherin under-
goes processing by c-secretase to generate an epsilon
cleavage product, N-Cad ⁄ CTF2, which forms a complex
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1871
in the cytoplasm with the transcription factor CBP, pro-
moting CBP ubiquitination, proteasomal degradation,
and repression of CBP ⁄ CREB transcription [63].
The PA-TM-RING proteins GRAIL [15], Goliath-
related E3 ubiquitin ligase 1 [64], h-Goliath [57] and

RNF13 [21] have been established to be E3 ubiquitin
ligases. Plant AtRMR1 has not been shown to have
this enzymatic activity. Conserved recognition motifs
on E3 target substrates have not been defined, making
detection of E3 substrates difficult. Only two substrates
of a member of the PA-TM-RING family have been
identified; RhoGDI [65] and CD40 ligand [66] have
been shown to be ubiquitinated by GRAIL. Identifica-
tion of the RNF13 substrate(s) will allow us to deter-
mine whether proteolysis is a positive or negative
regulator of RNF13 E3 activity.
In addition to PA-TM-RING proteins, only one
other family of mammalian integral membrane RING
E3 ubiquitin ligases has been identified that does not
localize to the ER. These proteins, members of the
membrane-associated RING-CH family [67], are
human homologs of viral immune evasion proteins
that downregulate cell surface glycoproteins, including
immune recognition molecules [68]. They possess
two TM domains and are structurally unrelated to the
PA-TM-RING proteins.
RNF13 was previously identified in a screen for
proteins whose ectopic expression stimulates neurite
outgrowth of PC12 cells cultured on collagen [22]. In
further support of a role for RNF13 in neuronal devel-
opment, we have determined by quantitative real-time
RT-PCR analysis that endogenous RNF13 is upregu-
lated upon induction of neurite outgrowth in B35 neu-
roblastoma cells. The RNF13 gene is thus responsive
to signaling cascades that result in cell differentiation

and neurite outgrowth. Although RNF13 homologs
have been identified in humans, dog, chicken, fruit fly,
mosquito, tobacco and rice, no PA-TM-RING protein
has been identified in yeast, suggesting that this E3
activity may be necessary to modulate a function
unique to multicellular organisms, such as cell–cell
interaction or tissue development.
Experimental procedures
Reagents
Bovine fibronectin, dibutyryl-cAMP, chondroitinase ABC,
MG132 and bafilomycin A1 were obtained from Sigma-
Aldrich (St Louis, MO, USA). Lipofectamine 2000 was
from Invitrogen (Carlsbad, CA, USA) and FuGENE 6
transfection reagent was from Roche Diagnostics (Indiana-
polis, IN, USA). Bicinchoninic acid reagents were from
Pierce/Thermo Fisher Scientific (Rockford, IL, USA). Flu-
orsave was from Calbiochem/EMD Chemicals (Gibbstown,
NJ, USA). Purified ubiquitin was a gift from J. McCarville
(UNC-CH). Rabbit E1 enzyme and human UbcH5a,
UbcH5c and UbcH6 E2 enzymes were from Boston
Biochemicals (Cambridge, MA, USA).
Antibodies
Affinity-purified polyclonal rabbit anti-RNF13 serum was
prepared by GenScript Corp (Piscataway, NJ, USA). using
the peptide CPNGEQDYNIANTV, the 14 C-terminal resi-
dues of mouse RNF13. Mouse and rabbit anti-FLAG IgG
sera, horseradish peroxidase (HRP)-conjugated anti-FLAG
M2 IgG1 serum and HRP-conjugated anti-HA IgG1 serum
were from Sigma-Aldrich. Sheep anti-mouse HRP-conju-
gated serum was from Amersham/GE Healthcare (Chalfont

St Giles, UK). Mouse anti-LAMP2 IgG2A and anti-CD63
IgG1 sera were from the Developmental Studies Hybrid-
oma Bank, University of Iowa. EEA1 IgG1 antibodies were
a gift from J. Trejo (University of California, San Diego,
CA, USA). Mouse anti-human Golgin 97 IgG1 serum,
mouse anti-human MPR Ig2A serum, donkey anti-mouse
AlexaFluor 488 IgG serum, donkey anti-rabbit Alexa-
Fluor 488 IgG serum and goat anti-mouse AlexaFluor 568
IgG serum were from Invitrogen. Goat anti-rabbit Texas
Red serum was from Jackson Immunoresearch (West
Grove, PA, USA). Anti-calnexin IgG serum was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA)
and anti-PDI IgG2A serum from Affinity Bioreagents/
Thermo Fisher Scientific (Rockford, IL, USA).
Expressed sequence tags, cloning, and
site-directed mutagenesis
Image clone 4317972 was obtained from ATCC and deter-
mined to encode complete RNF13 by sequencing. For
mammalian transient expression, RNF13 was cloned into
pCDNA3 (Invitrogen) and pSG5X (Stratagene, Cedar
Creek, TX, USA), pSG5 modified to contain an expanded
multiple cloning region. Mutagenesis was performed using
a QuikChange Site-Directed Mutagenesis kit (Stratagene)
according to the manufacturer’s instructions. Transiently
expressed RNF13 was mutated to possess a 1· FLAG
epitope inserted after residue 377. This site was initially
chosen because mouse RNF13 terminates with a valine,
and C-terminal valines have been shown to modulate tar-
geting of some integral membrane proteins [69–71]. Stably
expressed RNF13 was subsequently engineered to express

a C-terminal 3· FLAG tag added by cloning into p3·
FLAG vector (Sigma-Aldrich). The expression and target-
ing of the protein with the 3· FLAG epitope appeared to
be indistinguishable from those of the protein modified
with a 1· FLAG epitope inserted after residue 377
(Fig. 2). The oligonucleotides described in Table S1 were
Proteolytic regulation of RNF13 J. P. Bocock et al.
1872 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
purchased from Integrated DNA Technologies (Coralville,
IA, USA), Invitrogen, and the Nucleic Acids Core
Facility, UNC-CH. Automated sequencing was performed
at the UNC-CH Genome Analysis Facility.
Bacterial expression and purification
BL21DE3 bacteria were transformed with pET3E-His
vectors encoding mouse RNF13D1–205 or RNF13D1–205
with a C266A point mutation. Concentrations of purified
protein were determined using a bicinchoninic acid assay
according to the manufacturer’s instructions. Purified
protein was resolved on both 12% and 15% polyacrylamide
gels to assess purity; 15% gels were analyzed by Coomassie
stain, and 12% gels were western blotted with anti-6· His
serum. Relative amounts of protein were determined by
using densitometry software (kodak 1d, version 3.6.2,
Eastman Kodak, Rochester, NY, USA).
In vitro ubiquitin ligase assay
To perform the ubiquitin ligase assay, we used a reaction
mixture consisting of 50 mm Tris ⁄ HCl (pH 7.6), 2 mm
MgCl
2
,2mm ATP, 1 mm dithiothreitol, 100 nm rabbit E1

enzyme, 1 lm E2 enzyme, 100 ngÆlL
)1
purified ubiquitin,
and 0.1 lgÆlL
)1
purified RNF13. These mixtures were incu-
bated for 2 h at 25 °C. Samples were resolved on 12%
polyacrylamide gels, and western blots were probed with
anti-ubiquitin serum.
Viral expression
For stable expression, cDNA encoding epitope-tagged
RNF13 was cloned into the lentiviral FG12 vector [72].
B35 rat neurons [51], PC12 rat adrenal medulla pheochy-
romocytoma cells, human HeLa cells or CHO cells were
infected as previously described [72]. Green fluorescent pro-
tein-expressing cells were selected by fluorescence-activated
cell sorting by the Flow Cytometry Facility, UNC-CH, to
generate the cell lines CHO-RNF13-HAF, PC12-RNF13-
HAF, HeLa-RNF13-HAF and B35-RNF13-HAF, which
stably express RNF13, or CHO-Con (control) and B35-
Con, which stably express empty vector.
Cells and drug treatments
Mouse cortical neurons, prepared as previously described
[73], were a gift from L. Brennaman, UNC-CH. HeLa and
CHO cells were transfected using Lipofectamine 2000, and
COS cells were transfected using FuGENE 6, according to
the manufacturers’ instructions. When specified, cells were
treated for 8–12 h with dimethylsulfoxide (0.1%), the
peptide aldehyde MG132 (5 lm in dimethylsulfoxide), or
epoxomicin (500 nm in dimethylsulfoxide). NH

4
Cl (10 mm)
was added for 24 h, and bafilomycin A1 (1 lm) for 8 h.
When specified, RNF13 was immunoprecipitated from cells
and resolved on polyacrylamide gels as described previously
[46].
High-mannose sugar addition was inhibited by treatment
of cells with tunicamycin (Roche) at 1.5 lgÆ mL
)1
for 14 h.
N-linked sugar was removed by treating immunoprecipi-
tates with PNGase F (Roche and NE BioLabs, Ipswich,
MA, USA) according to the manufacturers’ instructions.
Chondroitinase
Immunoprecipitated protein eluted from resin in
SDS ⁄ PAGE loading buffer was divided into two equal
parts. After addition of pH 7 sodium acetate to a final con-
centration of 50 mm, chondroitinase ABC (0.2 U) or an
equivalent volume of SDS ⁄ PAGE buffer was added as
specified. Samples were incubated at 37 °C overnight. Dith-
iothreitol (50 mm) and bromophenol blue were added to
each tube, and proteins were resolved by 12% SDS ⁄ PAGE
and visualized by western blotting as indicated.
Cell fractionation
Cell fractionation was performed as described previously
[46], with addition of 10 mm sodium fluoride, 10 mm
sodium vanadate and 10 mm sodium pyrophosophate to
the homogenization buffer. Soluble cytoplasmic proteins
and microsomes were separated by centrifugation for 1 h at
60 000 g and 4 °C in a Beckman TL-100 centrifuge. The

pellet or microsome fraction contained all cellular mem-
branes. Microsomes were lysed, and the peripheral proteins
were stripped from the membranes by resuspending intact
microsomes in carbonate buffer containing 100 mm sodium
carbonate (pH 11.5), 100 mm KCl, and 5 mm EDTA. Frac-
tions were resuspended in SDS ⁄ PAGE buffer for analysis
using a Novex 4–12% Bis–Tris gel (Invitrogen) in Mops
running buffer (Invitrogen).
SDS
⁄
PAGE and immunoblotting
Cell monolayers were harvested by scraping into hot gel
loading buffer. Protein concentrations were determined
using a bicinchoninic acid assay according to the manufac-
turer’s instructions. Equal quantities of cellular protein
were routinely loaded in each lane of a given gel, as speci-
fied. Proteins were resolved on 12 · 14 cm 1-mm-thick
SDS-containing polyacrylamide gels of the percentage indi-
cated. Immunoblots were blocked with 1% nonfat dry milk
at either room temperature for 1 h or at 4 °C overnight,
and then probed with primary antibodies, either HRP-con-
jugated anti-FLAG M2 serum or HRP-conjugated anti-HA
serum. Western blots were developed using enhanced
chemiluminescence.
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1873
Confocal immunofluorescence microscopy
Immunofluorescence was performed as described previously
[74]. Confocal images were obtained with a Zeiss LSM 210;
images were assembled using adobe photoshop ele-

ments 2.0 and adobe illustrator.
Quantitative real-time RT-PCR analysis of RNF13
mRNA expression
Total RNA was harvested from adult and embryonic
mouse tissues and from cultured cells using TRIzol reagent
(Invitrogen) according to the manufacturer’s instructions.
Embryonic mice tissues were harvested at embryonic days
14.5 and 16.5. B35 neuroblastoma cells were cultured on
dishes coated with 5 lgÆmL
)1
fibronectin, and induced to
extend neurites by incubation with 100 lm dibutyryl-cAMP
for 3 or 5 days [51]. Quantitative real-time RT-PCR was
performed using the ABI PRISM 7700 Sequence Detection
System. RNF13 mRNA was normalized using 18S rRNA
as an internal control. The sequences of the oligonucleo-
tides and probes used are given in Table S2. Probes were
synthesized with a 5¢-FAM dye. These oligonucleotides and
probes were designed and synthesized by the UNC-CH
Animal Clinical Chemistry and Gene Expression Laborato-
ries. RNF13 oligonucleotides were used at 0.1 lgÆlL
)1
; 18S
oligonucleotides were used at 20 ngÆlL
)1
. RNF13 and 18S
probes were used at 20 lm. One hundred nanograms of
RNA was used for all reactions. The cycling parameters
used were as follows: 48 °C for 30 min, 95 °C for 10 min,
and 40 cycles of 95 °C for 15 s, followed by 60 °C for

1 min. Multiple independent samples were analyzed in
duplicate reactions for each data point. Data were analyzed
using api prism 7700 software. Changes in gene expression
were calculated using the 2
DDCT
method [75], with standard
errors derived from the generalized estimation equation
approach, which takes the potential correlation among
repeated measurements of duplicate reactions for each data
point into consideration [76].
Acknowledgements
We wish to thank V. Mauro, The Scripps Research
Institute, CA, and M. Lomax, University of Michigan
Medical School, MI, for generously sharing reagents
that were helpful in the initial stages of this work.
L. Brennaman kindly provided primary mouse neu-
rons. Microscopy was performed at the University of
North Carolina in the Microscopy Services Labora-
tory, Department of Pathology and Laboratory Medi-
cine, under the direction of C. Robert Bagnell Jr, or in
the Microscopy Facility, Department of Biology, under
the direction of T. Perdue. Quantitative real-time
RT-PCR was performed at the University of North
Carolina Animal Clinical Chemistry and Gene Expres-
sion Laboratories, UNC-CH, which is under the direc-
tion of H S. Kim. Cell sorting was performed at the
UNC-CH Flow Cytometry Facility, which is under
the direction of L. Arnold. This work was supported
by research grant MCB-0235680 (to A. H. Erickson)
from the National Science Foundation. S. Carmicle is

supported by NIH SPIRE Postdoctoral Fellowship
GM000678.
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Supporting information
The following supplementary material is available:
Fig. S1. tcoffee alignment [32] of murine RNF13
(mRNF13) (NCBI Locus AAH58182) with PA-TM-
RING proteins murine GRAIL (mGRAIL) (NCBI
Locus NP_075759) and A. thaliana (AtRMR1) (NCBI
Locus AAF32326).
Fig. S2. RNF13 has ubiquitin ligase activity in vitro.
Table S1. Oligonucleotides used for cloning RNF13
into vectors, introducing mutations and deletions, and
adding epitope tags.
Table S2. Oligonucleotides used for quantitative real-
time RT-PCR.
Table S3. Statistical analysis of RNF13 expression
data quantitated by real-time RT-PCR.

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J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1877