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

The C-terminal half of the protein contains a RING domain that func-tions as an ubiquitin ligase in vitro [1,2].. Keywords cellular targeting; endosomes; GRAIL; inner nuclear membrane; p

Trafficking and proteolytic processing of RNF13, a model PA-TM-RING family endosomal membrane ubiquitin ligase Jeffrey P Bocock1, Stephanie Carmicle2, Mayukh Sircar1and Ann H Erickson1

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 treatupregulat-ed 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: ann_erickson@med.unc.edu

(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.

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.

Dimeth ylsulf

oxide MG132 Epo xomicin

Dimeth ylsulf

oxide

MG132

1 2 3 4 5 6

72

36

55

kDa-Full-Length

+ Chondroitin sulfate

CTF-43

ICD

CTF-39 CTF-37

Epo xomicin

B

A

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 lM MG132 (lanes 2 and 5) or 0.5 lM 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].

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.

A

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 lM 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 lM 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 (lamin B) IgG, followed by donkey AlexaFluor 488 anti-goat serum.

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

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

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

Golgi

C N

RE

C N

EE

C N

ONM

INM

Nucleus

PM

Fig 5 RNF13 targeting pathways.

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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