(1) Destination Inner Nuclear Membrane

8/10/2019 (1) Destination Inner Nuclear Membrane

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Destination: inner nuclear membraneSantharam S. Katta1*, Christine J. Smoyer1*, and Sue L. Jaspersen1,2

1Stowers

Institute

for

Medical

Research,

Kansas

City,

MO

64110,

USA2Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA

The inner nuclear membrane (INM) of eukaryotic cells is

enriched in proteins that are required for nuclear struc-

ture, chromosome

organization,

DNA

repair,

and

tran-

scriptional control. Mislocalization of INM proteins is

observed in a wide spectrum of human diseases; how-

ever, the mechanism by which INM proteins reach their

final destination is poorly understood. In this review we

discuss how investigating INM composition, dissecting

targeting pathways of conserved INM proteins in multi-

ple systems and analyzing the nuclear transport of vi-

ruses

and

signaling

complexes

have

broadened

ourknowledge of INM transport to include both nuclear

pore complex-dependent

and

-independent

pathways.

Thestudy of these INM targeting pathways is important

to understanding nuclear organization and in both nor-

mal and diseased cells.

INM

proteins

and

nuclear

organization

The defining

feature

of

eukaryotes

is

the nucleus. Unlike

other organelles, thenucleus is formed bytwo lipid bilayers:

an

outer

nuclear membrane

(ONM) and an

INM

separated

by

a

lumen. The

ONMand INM

merge

at

discrete regionsof

the nuclear envelope (NE) where nuclear pore complexes

(NPCs)

reside (Figure

1).Composedof30 subunits present

in

multiple

copies,

NPCs

are considered to

be

gatekeepers

that restrict passage

of

macromolecules

into and out of

the

nucleus in all eukaryotes [1,2]. Structural and molecular

analysishas

shown

that theNPC

containsa

centralchannel

lined

with

phenylalanine-glycine

(FG)-rich

repeat-contain-

ing nucleoporins (FG-Nups), which are thought to limit

diffusion

of

molecules

with a

Stokes

radius

greater than

2.63 nm or a molecular weight of 4060 kDa [3]. The

NPC also contains a series of peripheral channels thatmay

play

a

role in

the transport

of

INM

proteins

(Figure 1; see

below) [46].

The ONM is contiguous with the endoplasmic reticulum

(ER) whereas the INM

is

distinct,

containing several

hundred to possibly a thousand proteins [7,8]. Studiesof

a

handful of

INM

proteins, including

the conserved

SUN (for Sad1UNC-84 homology) and LEM (for Lap1

emerinMAN1)

families,

have

revealed crucial roles for

INM

proteins in nuclear

structure,

organization,

and po-

sitioning (reviewed in [912]). Onemajor function of SUN

proteins is to

connect the nucleus to

the cytoplasmic

cytoskeleton

throughtheirinteraction

withONM

proteins

that bind to actin, dynein, microtubule-organizing cen-

ters,

or

intermediate

filaments. Several

SUN proteins as

well as LEM domain-containing proteins also

serve as

scaffolds to cluster nuclear factors involved in transcrip-

tional control, DNA

repair, and meiotic recombination

[11,13,14]. In metazoans, the function of SUN and LEM

proteins in nuclear organization is partially dependent onlamins andother lamin-associated proteins, which forma

NE-associated meshwork that is important for maintain-

ing the structural

integrity

of

the nucleus and the distri-

bution of NPCs [1518]. A myriad of human diseases,

ranging from

tissue-specific diseases of muscle,

brain,

bone, and fat to

multisystemdisorders

suchas the prema-

ture aging syndrome Progeria, are associated with muta-

tions in

the genes encoding lamins and INM components

[16,1921]. The etiology is unclear in many cases, but

studies using tissue-culture cells, mouse, Caenorhabditis

elegans, andDrosophilamodelshave revealed interdepen-

dence

between many INM proteins and lamins

for

their

localization

and/or

function

(e.g.,

[2227]).

Understandinghow INM proteins are properly targeted and how their

distribution

is regulated in

different

cell types

or

under

conditions such as stress

or

development

is crucial to

elucidate the mechanism of these disorders.

Soluble cargo and the NPC

The

transport

of

soluble cargos across the NE

has

been

extensively

studied

in

many

eukaryotic

systems and a

general set of principles for nucleocytoplasmic exchange

has been

established [1,28]. Trafficking of

cargos into and

out

of

thenucleusrequires

targeting

information

in

theform

of

a

nuclear localization sequence

for

entry

(NLS;

typically

a

short

basic

sequence or

two basic

sequences

separated by

a linker)or anuclearexport sequence forexit (NES;typically

a

short

stretch

of

hydrophobic

residues)

[29].

These

sequences are recognized by karyopherins (also known as

importins

and exportins)

which

facilitate movement

through thecentralNPC

channel. The

ability

of

karyopher-

ins to bind to their cargo depends on the smallGTPase Ran

(Ras-relatednuclearprotein).

A

gradient

of

RanGTP

in

the

nucleus

and RanGDP

in

the cytoplasm

facilitates the

binding and release of karyopherins and cargos, and it

generates the directionality

of

transport

(Figure

2). Al-

though there is some diversity in import andexport signals,

and

often

redundancy

between

the karyopherins,

these

basic properties can account for transport of individual

Review

0962-8924/$ see front matter

2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.10.006

Corresponding author: Jaspersen, S.L. ([email protected]).

Keywords: inner nuclear membrane; nuclear transport; NPC; SUN protein; LEM

domain.*These authors contributed equally to this work.

Trends in Cell Biology, April 2014, Vol. 24, No. 4 221
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proteins

andlargeprotein

complexesas

well

as

RNAs,which

are typically exported as part of a protein complex contain-

ing the targeting information

[2830].

It has

been widely assumed that the localization of

INM

proteins would follow similar principles to those governing

the localization

of

soluble

cargo.The

protein

might

diffuseor

be trafficked to the INM by an INM protein-derived target-

ing sequence in an NPC-dependent pathway. This is partic-

ularly true in fungi that undergo a closed mitosis where

NPC-mediated

transport

is

the sole

mechanism of

macro-

molecular

exchange between the nucleus and cytoplasm.

Therefore, it was a surprise when studies of several con-

served

INM

proteins

failed to

provide

a

simpleparadigm

for

INM

localization.

Belowwe

integrate ourknowledge of

INM

trafficking and discuss growing evidence for at least four

types

of

transport

pathways.

The

ongoing study

of

these

pathways

will

result

in

greater

understanding

of

the nucle-

us, its evolution, and its function in genome organization

and human

diseases

linked to

NE

dysfunction.

Diffusion-retention

The journey for a protein to the INM begins at the ER

where

the

integral

membrane

protein

is

co-translationally

or post-translationally

inserted

into

the

ER

membrane.

Similarly to other integral membrane proteins found

throughout

the

cell,

insertion

of

INM

proteins

into

the

ER

membrane

likely

involves

the

Sec61

translocon

[31,32]. Because the ER is contiguous with the ONM,

INM

proteins

are

thought

to

freely

diffuse

through

the

ER to the ONM. Consistent with this hypothesis, photo-

bleaching studies designed to assay the mobility of several

INM proteins showed rapid diffusion from the ER to the

NE

[3337].

The

observation

that

their

mobility

decreased

significantly

once

localized

to

the

INM

destination

sug-

gests that they are associated with relatively immobile

components

of

the

nucleus

such

as

lamins

or

chromatin.

Careful

examination

of

NPC

ultrastructure

reveals

that

its peripheral channels might be sufficiently large

(10 nm) to accommodate the diffusion or transport of

an

integral

membrane

protein

assuming

it

has

a

small

nucleoplasmic, or extralumenal, domain [6,38,39]. Studies

of

lamin

B

receptor

(LBR),

a

multispanning

INM

compo-

nent,

showed

a

strong

size-selection

during

INM

transport:

a

version

of

LBR

that

had

a 22 kDa extralumenal region

localized to the INM whereas a 70 kDa version did

not

[40,41]. Similar

studies

on

other

INM

proteins

also

Yeast

Metazoans

Ndc1 Ndc1

GP210

Pom34

Pom152

Pom33

TMEM33

Pom121

Transmembrane Nups

Yeast

Metazoans

Nup60

Nup1

Nup153Nup2 Nup50

Mlp1/Mlp2 Tpr

Nuclear FG Nups and basket

Yeast

Metazoans

Nup120

Nup160

Nup133

Nup133

Nup145C Nup96

Nup84

Nup107

Nup85

Nup85

Seh1

Seh1

Sec13

Sec13

Nup37

Nup43

Cdc31

Centrin-2

Aladin

Outer ring Nups

Yeast Metazoans

Nup82

Nup88

Nic96

Nup93

Nup188/192 Nup188/205

Nup157/170 Nup155

Nup53/59 Nup35

Inner ring and linker Nups

Yeast

Metazoans

Nup159 Nup214

Nup358

Nup42

hCG1

Nup82

Nup88

Gle1

Gle1

Cytoplasmic FG Nups and

filaments

Yeast Metazoans

Nup100/116/

145N Nup98/96

Nsp1 Nup62

Nup57 Nup54

Nup49 Nup58/45

Central FG Nups

Central channel

50 nm

Nucleoplasm

Peripheral

channels

10 nm

TRENDS in Cell Biology

Cytoplasm

Figure 1.

Schematic of the nuclear pore complex (NPC) showing subunits required for inner nuclear membrane (INM) targeting in yeast andmetazoans. Biochemical and

genetic analysis of theNPC shows that it is a modular structure. Its subunits, the nucleoporins (Nups), can be assigned to distinct functional subcomplexesbased on their

physicaland genetic interactions with other Nups.Poremembraneproteins (Poms)connect to outer ringNups to anchor theNPC in thenuclearenvelope (NE). Linker Nups

connect the outer ring to the inner ring Nups, and FG-Nups [phenylalanine-glycine (FG)-rich repeat-containing nucleoporins] form the central channel. Asymmetrically

localized Nups form thenuclear basket and thecytoplasmicfibrils (basedon [46]). Based on cryo-electron microscopymeasurements of thehuman NPC, thediameters ofthe central andperipheral channelsare approximately 50 and 10 nm, respectively [6].

Nups that have been testedfor a role in INMtrafficking arecolored, and those that are

required for transport of one or more INM proteins are in bold [37,43,48,51,52,84,85] .

Nups in gray have not been assayed for a role in INM trafficking.

Review Trends in Cell Biology April 2014, Vol. 24, No. 4

222


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indicate

that

there

may

be

an

upper

size-limit

on

transport

and have

shown

further

roles

for

lamins

as

well

as

chro-matin in

the

retention

of

proteins

at

the

INM

[35,37,42

44]. This biphasic state of fast- and slow-moving pools of

INM

proteins

associated

with

the

ER

and

INM,

respective-

ly,

combined

with

a

strong

size

selection

in

the

cargo,

suggest that an INM protein is synthesized on the ER,

diffuses

freely

between

ONM

and

INM

via

the

NPC,

but

is

preferentially

retained

at

the

INM

due

to

interactions

with

chromatin and/or lamins (Figure 3). The appealing sim-

plicity

of

this

model,

referred

to

as

diffusion-retention,

may

explain why it has predominated in the field until recently.

LEM domain proteins: active transport?

At

first

glance,

nuclear

trafficking

of

LEM

domain-contain-

ing

proteins

such

as

Man1

(LEM

domain-containing

pro-

tein 3), Lap2b (lamina-associated polypeptide 2b), and

emerin

appears

to

follow

the

pattern

of

the

diffusion-re-

tention

model

(Table

1) [33,35,36,42,45]. For

example,

the

localizations of human Man1 and Lap2b are largely de-

pendent

on

the

size

and

nuclear-binding

function

of

their

extralumenal

domains

(Table 1).A careful

dissection

of

the

N-terminus of Lap2b showed that it contains distinct

chromatin-

and

lamin-binding

domains;

however,

only

the

lamin-binding

region

is

required

for

INM

localization

[42,45].

Similar

results

were

also

obtained

for

theDrosoph-

ila LEM proteins Otefin, Bocksbeutal, and Man1 [46,47].

It

was

therefore

a

surprise

when

analysis

of

the

budding-yeast

LEM

domain-containing

proteins

Heh1

(he-

lix-extension-helix-1)/Src1

and Heh2

showed

that their

transport

is

not based on

diffusion-retention

but

instead

requires active transport similar to the pathway used by

soluble

cargos (Figure

4, Table 1) [48].

King

and colleagues

demonstrated

that the accumulation

of

Heh1YFP

and

Heh2YFP at the INM is dependent on karyopherin-a

(Kap60) and karyopherin-b (Kap95)

together

with

theRan GTPase cycle. They also identified a putative NLS in

Heh2

and showed

that this region (which includes

adjacent

residues) binds

to

karyopherins

and is

important for INM

localization. Based on their work, the authors proposed a

transport factor-based

model

for

INM

trafficking

(Figure 3)

[49]. Analysis of the primary sequence of other INM compo-

nents revealed that many

contain putative NLSs

in

their

extralumenal

domains,

suggesting

that this could be

a

widely used pathway for INM localization (Table 1) [49,50].

If

Heh1

and

Heh2

use

active

transport

and

require

the

same

transport

factors

as

soluble

proteins,

do

they

also

use

the central channel of the NPC for trafficking or are

peripheral

channels

used?

In

yeast,

as

in

higher

eukar-

yotes, the size of the peripheral channels is small andwould

probably

only

accommodate

a

protein

complex

with

a mass of2540 kDa [4,5,49,51]. It is therefore difficult to

imagine

how

an

integral

membrane

protein

bound

to

a

karyopherin-a/karyopherin-b complex

would

physically

fit

in the peripheral channel. However, it is also unclear how

an INM

protein

might

utilize

the

larger

central

channel,

which

is

located

in

the

center

of

the

NPC

approximately

50 nm away from the membrane region [6]. To test if Heh2

traverses

through

the

central

channel,

Meinema et al.

developed

a

clever

strategy

to

trap

translocation

inter-

mediates [52]. The authors constructed a synthetic INM

protein

by

fusing

the

Heh2

NLS,

its

linker

(L,

the

region

between

the

NLS

and

transmembrane

domain)

and

trans-membrane

domain

to

human

FKBP12

and

GFP

(FKBP

GFPNLSLTM). This was expressed in cells containing

a

version

of

the

central

channel

NPC

protein

Nsp1

(nucleoskeletal-like

protein/nucleoporin)

fused

to

the

FRB (FKBP12/rapamycin-binding) domain of human

mTOR

(mechanistic

target

of

rapamycin).

In

the

presence

of

rapamycin,

FRB

and

FKBP12

will

bind

if they

are

in

closeproximity [53]. If theHeh2NLSlinker constructuses

the

central

NPC

channel

during

transport,

it

should

be

possible to trap the synthetic INM protein in the NPC by

addition of rapamycin. If it uses peripheral channels,

rapamycin should have no effect because Nsp1FRB and

FKBPGFPNLSLTM

will

not

come

into

physical

prox-

imity.

NE

aggregation

of FKBPGFPNLSLTM

in

Nsp1FRB cells in a rapamycin-dependent manner was

observed,

consistent

with

the

idea

that

Heh2

uses

the

NPC

central

channel

[52]. The

fact

that

combinations

of FG

Nups when deleted also affected transport further sup-

ported

this

possibility

[52]. Curiously,

trapping

of

the

synthetic

INM

protein

did

not

affect

the

transport

of solu-

ble cargos but did affect the NE accumulation of additional

FKBPGFPNLSLTM,

suggesting

that

trafficking

of

INM

proteins

is

specifically

blocked

[52,54]. Thus,

it

appears

that

although

INM

and

soluble

transport

path-

waysmay at leastpartially overlap, there aredifferences in

the

actual

transport

mechanism.

Dedicated

NPCs

and/or

Karyopherin Karyopherin

NLS

Cargo protein

RanGTP

RanGDP

Ran GEF

Ran GAP

NES

Cargo protein


Figure 2.

Transport of soluble cargos. Targeting to the nucleus involves a nuclear

localizat ion sequence (NLS) that is recognized by an import-competent

karyopherin (also called an importin) that shuttles the cargo through the nuclear

pore complex (NPC) via its central channel. Binding of Ran (Ras-related nuclear

protein)GTP inside the nucleus causes the complex to disassemble. The

karyopherin can be recycled to the cytoplasm while the cargo accumulates in

the nucleus. During export, a nuclear export sequence (NES) is recognized by an

export-competent karyopherin (also called an exportin) together with RanGTP.

This ternary complex is transported through the NPC central channel to the

cytoplasm, where nucleotide hydrolysis is stimulated, causing RanGTP to be

converted to RanGDP, which releases the karyopherin and cargo. Abbreviations:

GEF, guanine nucleotide exchange factor; GAP, GTPase activating protein.


223


4/9

Diffusion retenon Transport factor mediated

Sorng mof mediated Vesicle mediated

Protein cargoProtein cargo

Protein

NLS

RanGTP

Ribosome

Sec 61

Nup 50/Nup 2

Imporn 16

Lamin or chroman binding domain

Peripheral channel

Lamins

Chroman

Karyopherin

Central channel

Protein

yt lasm

Nucleoplasm

yt lpasm

Nucleoplasm

t lpasm

Nucleoplasm

Cytoplasm

Nucleoplasm

R

INM-SM


Figure 3.

Four proposed INM targeting pathways. Integral membrane proteins are synthesized and inserted into the endoplasmic reticulum (ER) membrane either co-

translationally or post-translationally. (A) The diffusion-retentionmodel suggests that theinnernuclearmembrane(INM)protein is able to diffuse freely from theER to theouternuclearmembrane (ONM),and to diffuse from theONM to INMusing peripheral nuclear pore complex (NPC) channels. Accumulationof theproteinat theINMoccurs

due to tethering by chromatin and/or lamins. Because transport relieson peripheralchannels, theextralumenal domainmust be small (


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the requirement for INM proteins to stretch and dodge

between

the

NPC

components

may

partially

account

for

differences

in soluble

and

membrane-based

transportthrough

the

NPC.

The

INM

sorting

motif

Although

many INM proteins

contain

putative

NLSs,

it

is unclear in most cases if these are essential for INM

transport.

Often, their mutation

or

deletion

does

not

affect localization,

suggesting

that

additional features

contribute to INM targeting (e.g., [44,55]). The baculo-

virus

occlusion-derived virus integrates

into

the ER and

then traverses to the nucleus for viral envelope assem-

bly. Analysis of this pathway revealed two unique fea-

tures that are essential for its trafficking to the INM: a

hydrophobic

transmembrane sequence and

an

adjacent

region

containing positively charged amino

acids

such

as

lysine or arginine, termed the inner nuclear membrane

sorting motif

(INM-SM)

[56,57]. Owing to

the correlation

that

is known

to

exist

between

the length

and composi-

tion of transmembrane domains and the intracellular

location

and topology

of

the protein, this finding

may not

be surprising

[58].

However, the observation that the

INM-SM is present on many NE components, including

some

LEM domain- and SUN domain-containing

pro-

teins,

the INM proteins nurim and LBR, and the integral

membrane

NPC components, GP210 (nucleoporin

210 kDa) and Pom121 (nuclear pore membrane protein

121 kDa),

suggests

that

sorting

motifs

may play a

general role in targeting proteins to the INM

(Figure 3, Table 1) [56].

Consistent

with

the

idea

that

the

INM-SM

plays

anactive

role

in

INM

transport

rather

than

an

indirect

role

in

protein topology, the INM-SM from the baculovirus-de-

rived

protein

and

from

LBR

and

nurim

were

shown

to

bind

to

a

truncated,

membrane-associated

form

of karyo-

pherin-a termed importin-a-16 (or KPNA-4-16) [59,60].

The

smaller

importin-a-16 lacks

the

importin-b-binding

domain

that

normally

facilitates

karyopherin-a interac-

tion with karyopherin-b, suggesting that importin-a-16

functions

independently,

rather

than

as

part

of

a

dimeric

karyopherin complex [61]. The formation of a truncated

version of karyopherin-a is not due to the virus or the cell

line: karyopherin-a in insects, humans, and yeast is pres-

ent

in

multiple

isoforms

that

arise

through

alternative

splicing

and/or

internal

initiation

of

transcription

[59,60,62,63]. It was observed that importin-a-16 was

crosslinked

with

Sec61a at

the

ER

membrane,

leading

to

the

proposal

that

the

INM-SM

may

specifically

recognize

INM proteins as they emerge from the ribosome during

translation

to

facilitate

their

transport

to

the

INM

(Figure

3) [59,61,64].

Interestingly, the trafficking of Heh2 in yeast, which

was

originally

used

to

demonstrate

the

importance

of

transport

factors

in

INM

localization,

provides

an illustra-

tion

of

signal

sequence-based

transport.

The

observation

that the N-terminal region of Heh2 mediates binding to

Kap60

(the

yeast

karyopherin-a) in vitro, and that

Table

1.

Sequence

features

in

well-characterized

INM

proteins

and

their

roles

in

INM

transporta

Domainsb Size of the

extra-lumenal

domain (kDa)c

Nuclear localization

sequence (NLS)

Inner nuclear

membrane sorting

motif (INM-SM)

Golgi-retrieval

sequence

Other Refs

LEM domain

Emerin (Hs) 26 31-RRLYEKKIFEYETQRRR-46 211-RAPGAGLGGD-221 44-RRR-46 ATP-dependent [43]

Man1

(Hs) 50(Nt)/31(Ct) 190-RRKP193

285-RPRR-28

706-OGDRKKM-712

457-KREEVSPTGSFSAH-471 None Linker [35]

Lap2b

(Rn) 45 257-PRRRVEP-263 403-KTKK-406 251-RGSRR-255

258-RRR-260

[37,43]

Heh1 (Sc) 50(Nt)/13(Ct) 86-PRRSRRA-92

93-RREKSASPMAKQFKKNR-109

173-RKKRK-177

216-KKRK-219

433-KFKRALKFLSK-453

726-KNYRKK-734

None Linker [52]

Heh2

(Sc) 36(Nt)/12(Ct) 125-KKKRKKRSSKANK-137 302-KTKRGIDIMK-311 None Linker [48,52,54,63]

SUN domain

Sun1 (Hs) 27 None predicted None None SUN-NELS

209-SRVYSRDRNQK-219

[44,85,86]

Sun2 (Hs) 23 38-PLRTLKRKSSNMKR-52 None 102-RRRR-105 Sun domain,

ATP-dependent

[43,55,68,87]

UNC-84 (Ce) 59 35-KVRRK-39

170-HRRR-173

503-KKSSK-507 171-RRR-173 SUN-NELS 235-

SRMTTRSQTLER-246

[44]

Mps3 (Sc) 18 None 146-KKLK-150 None Htz1 [69,70]Other

LBR (Hs) 23(Nt)/25(Ct) 63-RKGGSTSSSPSRRRGSR-79

95-RRSASASHQADIKEARR-111

169-RPRR-172

252-RAKD-256 74-RRR-76

169-RPRR-172

Ran-dependent [41,43]

aFeaturesin bold havebeentestedin thecontextof thefull-lengthprotein andshownto playa rolein INMtranslocation,whereasfeatures in regulartypeappear tohavelittle

to noeffect on targeting.Other featureshave beenpredictedbased onamino acidcomposition, sequencehomology,or other methods,but their roles in transport havenot

beenanalyzed in the contextof the full-lengthprotein (see [44,49,56,88]).Note,onlyclassical NLSs havebeen predicted; othertypesof NLSs,suchas thePY-NLS,havenot

been identified in INM proteins.

bCe, Caenorhabditis elegans;

Hs, Homo sapiens;

Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae.

cCt, C-terminal; Nt, N-terminal.


225


6/9

transport

of

Heh2YFP

is

dependent

on

the function of

KAP60 aswell asKAP95 (karyopherin-b), implicated trans-

port factorsinHeh2 INM localization [48].However,further

analysis of

the karyopherin-a isoforms

required

for

Heh2

import

revealed that full-length

Kap60 is

not required fortransport

of

Heh2. Instead,

Kap60-44

or

Kap60-30

isoforms

are needed to enrich Heh2 at the nuclear periphery [63]. A

previously

unrecognized INM-SMin

Heh2

was

mutated

and

this

reduced

Heh2

NE

localization, indicating thattheINM-

SM contributes to its INM localization (Table 1). Details of

the

signal-sequence mechanism of

transport

remain

to

be

elucidated, including whether

Kap60-44

simply

delivers

Heh2 to the NPC and then a full-length Kap60/Kap95

complex forms,

or

whether

Kap60-44

functions

as

a

karyo-

pherin and shuttles Heh2 into the nucleus. Studies ofviral

protein transport suggest that the latter is true [64]; it will

therefore be interesting to determine if Kap60-44 uses the

peripheral

or

centralchannelandalsoto

dissecthow

Kap60-

44

disassociates

from

theHeh2

cargo once

inside

thenucle-

us. Full-length karyopherin-a releases its cargo in a Ran

GTP-independent

mechanism involving nucleoplasmic

Nups

(vertebrate

Nup50/yeast

Nup2)

or

other

complexes

required for nuclear export (vertebrate CAS/yeast Cse1)

[6567]. Nup2

is

one of

the two Nups

required for

transport

of Heh1YFP

and

Heh2YFP,

making

it

temptingto

specu-

late thatits involvementmaybe related tonuclear release of

Kap60-44 from

Heh1

or

Heh2

(Figures 1

and 3) [48].

SUN proteins: Golgi retrieval and redundant pathways

From extensive analysis of the LEM domain-containing

proteins,

LBR

and

various

viral

proteins,

it

is

clear

that

at

least three

modes

of

INM

localization

exist:

(i)

diffusion-

retention,

(ii)

transport

factor-,

and

(iii)

signal

sequence-

mediated transport. What is less well understood is the

relative

importance

or

dominance

of

one

pathway

over

another.

One

way

to

address

this

issue

is

to

study

thetransport

of

an

INM

protein

(or

a

class

of

INM

proteins)

that contains features necessary for all three modes of

targeting

and

systematically

eliminate

each

to

test

the

outcome

on

INM

localization.

Based

on

primary

sequence

information, the SUN family is an excellent model to test

the

requirement

of

different

trafficking

pathways

because

the

multiple

sequence

features

needed

for

INM

trafficking

are found in the N-terminal extralumenal domains ofmost

SUN

proteins.

For

example,

mammalian

Sun1

and

Sun2,

and yeast Mps3, all have small extralumenal domains,

Sun2 and C. elegans UNC-84 contain a NLS, and UNC-84

andMps3have INM-SMs (Figure4, Table 1) [44,55,6870].

To

determine

if diffusion-retention

is

important for

tar-

geting, thesize

of

the extralumenal

domain

can be

increased

byaddingone, two,or three copies ofGFP.When onecopy of

GFP

was

fused

to

the extralumenal

domain

of

Sun2 it

was

efficiently

transported

to

the NE,

but

adding two

or

three

copies of GFP virtually abolished transport [55]. A similar

size-dependence

was

also observed for

Mps3

[69].

These

results

suggest that diffusion-retention

is

important

for

INM localization of SUN proteins. However, the idea that

SUN

proteins

reach the INM

by

diffusion

is

problematic for

two

reasons.

First,

some

SUN

proteins

such

as

UNC-84have

large (59 kDa) extralumenal domains; it is unlikely that

they can reach the INM by simple diffusion. Second, and

more

compelling,

theN-termini of

Sun2, UNC-84, or

Mps3

Heh1 Heh2

Man1

MSC domain

INM-SM

SUN-NELS

LEM domain/

LEM-like domain

NLS

Key:

RRM domain

Coiled-coils and

SUN domain

Golgi retrival sequence

Lamin B tudor domain

Lap2

Emerin Sun2 Sun1

UNC-84

LEM domain SUN domain Other

Mps3LBR

Cytoplasm

Nucleoplasm


Figure4 .

Schematic showing theLEM (Lap1emerinMAN1) and SUN(Sad1UNC-84 homology) proteinsfrom different species as well as thelamin B receptor (LBR). The

approximate location of sequence features involved in inner nuclearmembrane (INM) localization and in other functions is indicated. For sequence coordinates,

see Table 1.

Other abbreviations: INM-SM, INM sorting motif; MSC,Man1Src1pC-terminal domain; NELS, nuclear envelope localization sequence; NLS,

nuclear localization sequence;

RRM,RNA

recognitionmotif..


226


7/9

are each

sufficient

to

target a

heterologous protein

to

the

INM

and to

prevent

its

diffusion back out of

the nucleus

[44,55,71]. Althoughan

argument

could

be

madethat this is

NLS-dependent, it is important to note thatMps3 does not

contain any recognizable NLS,

and removal

of

theNLSs

in

UNC-84 and Sun2 only

resulted in

minor

mislocalization

of

the corresponding full-length protein (Figure 4) [44,55,68].

Taken

together,

these data

suggest

that the N-termini

ofSUN proteins contain INM targeting information for path-

ways

other

than diffusion-retention

and transport factor-

mediated

trafficking.

Themost obvious candidate, the INM-SM, was tested in

both

yeast

and

worms.

In

the

case

of

Mps3,

the

INM-SM

had no apparent effect on localization or topology but its

mutation

did

result

in

defects

in

chromosome

organization

[70]. By

contrast,

mutation

of

the

INM-SM

in

UNC-84

resulted in a significant decrease in the level of the protein

at the

NE,

but

a

more

sensitive

assay

for

protein

function

at

the

INM

[rescue

of

the

nuclear

migration

defect

in hyp7

cells of unc-84(n369) animals] showed that at least some

mutant

protein

must

localize

because

migration

appeared

to be largely unaffected [44]. An additional motif identifiedby

virtue

of

its

homology

to

human

Sun1

(the

SUN-NELS

for SUN nuclear envelope localization sequence) was also

tested for

its

effect

on

UNC-84

localization

and,

like

the

INM-SM,

was

also

not

essential

(Figure 4, Table

1). Be-

cause this motif is not present in all SUN proteins, it likely

functions

in

conjunction

with

other

pathways,

possibly

as

a

non-canonical

NLS

or

in

a

piggy-backing

mechanism

simi-

lar to that required for Mps3 localization (Table 1) [44,69].

Consistent

with

this

idea,

only

when

the

INM-SM,

SUN-

NELS,

and

NLSs

were

mutated

did

UNC-84

mislocalize,

demonstrating that at least three, possibly four, INM

trafficking

pathways

must

be

blocked

to

abolish

INM

transport

in the

C.

elegans

system

[44]

(see

below).Because

Sun2

lacks

both

the

SUN-NELS

and

a

recog-

nizable INM-SM, a deletion analysis was conducted to

determine

regions

of its

N-terminus

required

for

INM

targeting.

This

unbiased

approach

uncovered

an

unlikely

motif: a cluster of arginine residues that functions as a

Golgi

retrieval

sequence

[55,72]. Only

when

this

region

was

mutated

in

combination

with

the

NLS

did

Sun2

disappear

from the INM. Interestingly, it was redistributed to the

Golgi

membrane,

leading

the

authors

to

search

for

sequences that might be involved in retrograde trafficking

from the Golgi to the ER. They found a Golgi retrieval

sequence and demonstrated that this domain was required

for binding

of

Sun2

to

components

of

the

coat

protein

complex

(COPI)

[73]. This

finding

suggests

that

accumula-

tion of proteins in the ONM/ER, perhaps by preventing or

recovering

the

protein

from

later

secretory

compartments,

may

be

a

requisite

step

in

INM

localization.

One

of

the

NLS

sequences that contributes to UNC-84 localization may in

fact

be

a

Golgi

retrieval

sequence,

indicating

that

Golgi

retrieval

could

be

a

conserved

aspect

of

sorting

SUN

proteins to the INM (Table 1) [44]. Because of the small

size

of

the

motif,

it

is

difficult

to

use

in

a

genome-wide

search,

but

it

is

feasible

to

test

individual

INM

proteins

for

groups

of

35

arginines.

Inspection

of

mammalian

emerin,

Lap2b, and LBR shows that all contain arginine-rich

regions

that

may

serve

as

Golgi

retrieval

sequences

(Figure

4, Table

1). The

role

of

these

residues

is

currently

unknown,

and

an

important

future

direction

is

to

deter-

mine their

role

in

INM

transport:

do

they

function

as

NLSs

and mediate karyopherin binding, or do they serve as Golgi

retrieval

sequences

and

play

a

role

in

binding

to

the

COPI

complex.

Non-canonical

pathways

for

INM

transportThe emerging picture of INM transport based on analysis

of

SUN

and

LEM

proteins

is

that

INM

localization

is

determined

by multiple cis- and trans-acting factors. A

systematic study of INM localization of 15 different INM

proteins

fused

to

GFP

and

expressed

in

transiently

trans-

fected HeLa cells reconfirms results obtained by previous

studies

of

individual

proteins

showing

that

diffusion-re-

tention,

transport

factors,

and

the

NPC

and

ATP

play

roles

in INM targeting of a subset of components (Table 1).

However,

this

study

clearly

illustrates

the

notion

that

INM

proteins

use

multiple

overlapping

pathways

to

reach

the INM, and suggests that additional unknown pathways

contribute

to

protein

localization

at

the

NE.

For

example,

the authors note that many INM proteins are enriched inFG-repeats,

and

propose

a

role

for

these

regions

as

trans-

port receptors for the protein that would aid in NPC

navigation

[43].

A

recent

study

of

Wnt

signaling

at

the

neuromuscular

junction in Drosophila larvae has also reinvigorated the

idea

of

non-canonical

NPC-independent

transport

mecha-

nisms

[74]. In

this

study,

nuclear

export

of

messenger

ribonucleoprotein (mRNP) granules harboring synaptic

transcripts

was

found

to

occur

through

a

vesicle-mediated

pathway

similar

to

viral

egress

used

by

herpes

virus

[75,76]. Although it is currently unclear if this type of

transport

is

widely

used

or

if

it

is

able

to

move

cargoes

from

the

ONM

to

INM,

it

suggests

that

novel

transportmechanisms

may

exist

(Figure 4). A

vesicle-mediated

INM

transport mechanism may help to explain curious genetic

interactions

observed

in

yeast

between

mutants

in

compo-

nents

of

the

vacuolar

sorting

complex

and

transcription

factors [7779].

A

comparison

between

the ONM and INM

of

the NE

and

the

membranes

of

mitochondria has

also led

to

theproposal

ofa threadingmodel for INM transport: theproteinwouldbe

spun througha

channel in

theONM into the lumenal space,

then threaded through another channel and released into

the INM (see [80,81]). Although there is little evidence to

support this model, it is worthwhile to consider that some

components

of

the Sec61

translocon

may

transiently

reside

at

the INM

as

well

as

the ONM/ER

because they are

required for the INM localization of growth factor receptors

such

as

the epidermal

growth factor

receptor [82,83]. Local-

ization of

components of

the ER-associated protein

degra-

dation machinery such as Doa10 to both the ONM/ER and

INM

also supports

the idea

that proteins

might

be

able exit

the

lumenal space into the nucleoplasm through a

mem-

brane translocation channel [51].

Concluding

remarks

Although

trafficking

of

proteins

and

macromolecules

in

and out of thenucleus is essential fornuclear maintenance,

growth,

and

proliferation,

the

mechanisms

for

travel,


227


8/9

especially

the

route

taken

by

INM

proteins

to

reach

their

final

destination,

appear

to

be

more

varied

and

complex

than previously

postulated.

Careful

dissection

and

analy-

sis of the targeting of each INM component is essential

because

multiple

pathways

converge

to

ensure

its

distri-

bution

to

the

INM.

It

is

currently

unclear

why

redundant

targeting mechanisms exist, although it is possible that

this

allows

tight

regulation

of

INM

content

in

developmen-tal-, tissue-, and cell cycle-specific ways. Because INM

proteins

affect

chromosome

organization,

transcriptional

regulation,

nuclear

morphology,

and

genomic

integrity,

dynamic control of INM materials may be necessary to

respond

to

signaling

pathways,

growth

and

environmental

cues, and to manage DNA damage. An exciting challenge

for

the

future

will

be

to

integrate

our

knowledge

of

these

different

areas

and

watch

the

INM

respond

in

real-time

to

various changes encountered by cells.

Acknowledgments

We thank Brian Slaughter and members of the Jaspersen lab for

discussion and comments, and Mark Miller for assistance will illustra-

tions. Support was

provided

by the Stowers

Institute for

MedicalResearch and the American Cancer Society (RSG-11-030-01-CSM).

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Documents

(1) Destination Inner Nuclear Membrane