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TICB 1254 No. of Pages 10
Review
Shaping the Endoplasmic
Reticulum into a Social
Network
Hong Zhang1,* and Junjie Hu1,*
In eukaryotic cells, the endoplasmic reticulum (ER) is constructed as a network
of tubules and sheets that exist in one continuous membrane system. Several
classes of integral membrane protein have been shown to shape ER membranes. Functional studies using mutant proteins have begun to reveal the
significance of ER morphology and membrane dynamics. In this review, we
discuss the common protein modules and mechanisms that generate the
characteristic shape of the ER. We also describe the cellular functions closely
related to ER morphology, particularly contacts with other membrane systems,
and their potential roles in the development of multicellular organisms.
Unique Morphology and Diverse Roles of the ER: A Perfect Model for
Connecting Membrane Dynamics to Organellar Functions
The ER is a single membrane-bound organelle involved in many critical cellular processes,
including protein synthesis, lipid synthesis, and calcium storage. Some organelles adopt
globular shapes, but the ER consists of interconnected membrane tubules and sheets [1,2].
From the center of a cell, the ER starts as the outer nuclear membrane (ONM), which is the outer
layer of the nuclear envelope (NE). Most of the ER sheets, which are cisternal structures with two
closely apposed membranes, appear in the perinuclear region. ER tubules, which typically form a
reticular network, exist in both the perinuclear and peripheral regions.
The distribution of ER sheets and tubules is tightly regulated in the cell. ER morphology can vary
substantially in different cell types or in response to different growth cues and conditions. Some
cells exhibit special ER arrangements. In yeast and plant cells, a large portion of the ER gathers in
the cortex; a region immediately beneath the plasma membrane [3]. The cortical ER usually
contains a tubular network with interspersed patches of sheets. Muscle cells contain a specialized ER, termed the sarcoplasmic reticulum (SR), which runs through the myofibril matrix as
tubules but merges into cisternal structures when engaging the plasma membrane [4]. In
addition, the ER of neuronal axons or plant root hairs is predominantly tubular, but professional
secretory cells, such as b-pancreatic cells or plasma cells, turn their ER into a massive stack of
sheets [5]. Thus, ER morphology is clearly linked to function.
How the characteristic shape of the ER is generated is a fundamental question in cell biology. Our
understanding of ER shaping began with the identification of tubule-forming proteins, namely the
reticulons (Rtns) and DP1/Yop1 [6] (Figure 1). The ER-bound dynamin-like GTPases atlastin (ATL)
and Sey1p/RHD3 were [7_TD$IF]then found to fuse ER membranes [7,8]; a key process in tubular network
formation. The tubule-forming proteins are also implicated in the generation of ER sheets, along
with another set of ER-resident proteins that include Climp-63, kinectin, and p180 [9]. Additional
candidates for regulating ER morphology are the Lunapark (Lnp) family [10–12], protrudin [13,14],
Rab10 [15], and Rab18 [16]. Although recent research has provided some mechanistic insight
Trends in Cell Biology, Month Year, Vol. xx, No. yy
Trends
ER sheets and tubules are generated by
membrane curvature stabilization, and
tubules are fused into a reticular network by membrane-bound GTPases.
Mechanistic studies reveal that transmembrane
hairpins,
amphipathic
helices and coiled coils are important
tools for ER morphogenesis.
The ER contacts other organelles at
discrete sites, where lipid transfer
and calcium exchange occur. Various
aspects of organelle dynamics, including fission, maturation and positioning,
are also regulated by formation of contacts with the ER. The morphology of
the ER controls the shape and area of
the contact with other organelles, and
thus the function of contact sites.
Formation and maintenance of ER
morphology are of general importance.
When ER morphology is perturbed,
organelle contacts, membrane trafficking and other ER functions are affected,
and neurodegenerative diseases may
result.
1
National Laboratory of
Biomacromolecules, Institute of
Biophysics, Chinese Academy of
Sciences, Beijing 100101, China
*Correspondence:
[email protected] (H. Zhang) and
[email protected] (J. Hu).
http://dx.doi.org/10.1016/j.tcb.2016.06.002
© 2016 Elsevier Ltd. All rights reserved.
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Rtns
Protrudin
ATL
Sey1p/RHD3
Kinecn
Glossary
Key:
RHD
Rtns
Reeps
ER tubule
Transmembrane
hairpin (TMH)
ER sheet
Climp-63
RHD
Amphipathic helix
(APH)
Coiled coil (CC)
Reeps
Lnp
p180
Figure 1. Determinants of ER Morphology. Endoplasmic-reticulum-shaping proteins are shown in either tubules or
sheets. Common protein modules, including transmembrane hairpins (TMH), amphipathic helices (APH), and coiled coils
(CC), are highlighted. Variable regions within protein families are shown by dotted lines. RHD, reticulon-homology domain,
formed by two TMHs with a connecting loop.
into these proteins, how the cell achieves the complex and dynamic morphology of the ER
remains a mystery.
Another important question is how the ER takes advantage of its morphological features. The
flattened surface of sheets is speculated to accommodate ribosome/polysome translation better
than tubules; thus, sheets are mainly involved in protein synthesis [9]. [8_TD$IF]By contrast, the curved
surface of tubules would be ideal for generating bent membranes, such as vesicles exiting the
ER [17]. Genetic alteration of regulators of ER morphology allows us to better understand the
correlation between ER shape and physiological function.
Mechanisms of ER Shaping and Remodeling
Formation of ER Tubules
ER tubules are cylindrical structures with a diameter of 30 nm in yeast and 50 nm in
mammals. Rtns were identified as ER tubule-forming proteins in an in vitro ER network formation
assay using Xenopus membrane extracts [6]. Subsequent analysis uncovered a similar protein
named REEP5/DP1 in mammals and Yop1p in yeast. Overexpression of these proteins results
in more ER tubules, and deletion or depletion has the opposite effect. The role of Yop1p or Rtn1p
in tubule formation was confirmed when purified reconstituted protein generated membrane
tubules in vitro [18].
The Rtns (four genes in mammals) and REEPs (six genes in mammals) contain a reticulonhomology domain (RHD), which is composed of two tandem hydrophobic segments. The length
of each hydrophobic segment (30–35 amino acids) allows [9_TD$IF]it to be embedded in a membrane
[10_TD$IF]most likely as a wedge-shaped hairpin. This configuration occupies more space in the outer
leaflet of a membrane bilayer than in the inner leaflet, curving the membrane. Searching
mammalian proteins based on structural conservation of the RHD predicted that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6IP1) and family with sequence similarity 134, member
B (FAM134B) contain similar tandem transmembrane hairpins (TMHs, see Glossary) [19]. Like
Yop1p, purified Arl6IP1 induces membrane tubule formation when reconstituted with lipids [19].
FAM134B has been proposed to regulate ER-phagy in addition to having membrane shaping
activity [20]. Thus, ER tubules can be formed and stabilized by a large set of proteins that share
a common domain.
In addition to inducing curvature, TMHs target proteins to ER tubules. Some TMHs traverse the
bilayer completely, but they are usually connected by just a few residues. These less asymmetric
hairpins do not actively bend membranes, but rather sense and prefer the presence of tubulelike membrane environments. TMH-containing proteins that localize to ER tubules include the
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Amphipathic helix (APH): an
/-helix with hydrophobic residues on
one side and hydrophilic residues on
the other side. These helices can
insert shallowly into membranes, in
some cases inducing curvature, and
in other cases sensing it.
Coiled coil (CC): a structural motif in
proteins where /-helices are
intertwined. Hydrophobic residues
are usually packed in the core of the
coil, and repeated sequences are
common. CCs are often involved in
oligomerization and tethering.
ER-mitochondria encounter
structure (ERMES): a complex
involved in ER–mitochondria contact
formation in yeast, comprising the
integral ER membrane protein Mmm1,
the cytosolic protein Mdm12 and two
mitochondrial outer-membrane
proteins (Mdm10 and Mdm34).
Extended synaptotagmins
(E-Syts): integral ER membrane
proteins involved in ER–PM tethering.
They contain a cytosolic
synaptotagmin-like mitochondrial-lipid
binding protein (SMP) domain
followed by multiple C2 domains. The
orthologous proteins in yeast are the
Tricalbins.
Oxysterol-binding protein (OSBP)related domain (ORD): a domain
found in OSBP and the closely
related OSBP-related proteins (ORPs)
(Osh proteins in yeast) that sense
sterols and/or transfer sterols at
contact sites. ORD binds cholesterol
and oxysterols.
Transmembrane hairpin (TMH):
connected transmembrane /-helices
that do not traverse membranes
completely, or have a very short
linker. TMHs are often found in
curved membranes, such as ER
tubules. They help to induce
membrane curvature or target integral
membrane proteins to highly curved
membranes. The reticulon-homology
domain is formed by tandem TMHs.
VAMP-associated proteins
(VAPs): integral ER proteins that
interact with tethering factors and/or
effectors via the FFAT motif (two
phenylalanines in an acidic trait) for
formation of membrane contacts.
VAPs contain a major sperm protein
(MSP) domain that binds to PI(4)P
and PS. Mammalian cells contain
VAP-A and VAP-B. Their yeast
orthologs are Scs2 and Scs22.
VAMP, vesicle-associated membrane
protein.
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ER fusogens ATL and Sey1p/RHD3, the microtubule-severing protein spastin, and the ER
morphology regulators Lnp and protrudin[1_TD$IF] (Figure 1).
Two mechanisms have been proposed for curvature generation in ER tubules by tubule-forming
proteins: the insertion of RHD wedges and scaffold formation via protein oligomerization [2].
Although these proteins clearly form homo- and hetero-oligomers [21], the molecular architecture of individual RHDs and precise mechanisms underlying oligomer assembly remain to be
investigated. Recent studies of Yop1p revealed that an amphipathic helix (APH) C-terminal to
its RHD is essential for tubule formation [22]. The helix is protected by lipids[2_TD$IF] but not detergent[3_TD$IF]
upon trypsin digestion[12_TD$IF] [18], and may insert into the membrane as an additional wedge.
Sequence analysis showed that the APH is conserved among most ER tubule-forming proteins.
In some family members[13_TD$IF], an APH is also the predicted N-terminal of the RHD. These RHDflanking elements provide additional stabilization.
ER tubule formation does not solely rely on Rtns and Rtn-like proteins. In mammalian cells, tubules
are constantly pulled out of the plane of ER membranes; an effect related to microtubule sliding
or tip binding [23]. The small GTPase Rab10 marks the growing tip of ER tubules [15]. Overexpression of a dominant-negative form or depletion of Rab10 causes ER sheet expansion. When
a microtubule-based force generates a new ER tubule, RHD-containing proteins are thought to
move in to stabilize it. Precisely how Rab10 regulates this process remains to be determined.
Formation of ER Sheets
ER sheets are cisternal structures with a constant distance between two apposed membranes.
Three mechanisms have been proposed for sheet formation [2] (Figure 1). First, membrane at
the edge of ER sheets is highly bent, with a curvature similar to a tubule cross-section. Thus, the
curvature of sheets and tubules is likely generated in the same way, possibly by the same
proteins. Tubule-forming proteins outline patches of ER sheets in cells, confirming their dual
roles in ER morphogenesis [9]. Second, the thickness of an ER cisterna is usually fixed,
suggesting the existence of a luminal spacer. Integral membrane protein Climp-63, which
localizes exclusively in sheets, is thought to use its luminal coiled coil (CC) domain to bridge
the two apposed membranes [24]. Consistent with its role in sheet formation, overexpression of
Climp-63 increases the number of ER sheets in cells [9]. Third, the surface of ER sheets is kept
flattened, likely by the sheet-enriched integral membrane proteins kinectin and p180 [9]. These
proteins possess a cytosolic CC domain, which may form rod-like scaffolds.
The NE, another sheet-like structure in the ER, may use the same mechanisms to generate and
maintain its shape. The ONM and inner nuclear membrane (INM) are connected at nuclear pores.
As the thickness of the NE is 26 nm, which is nearly half that of ER sheets, the curvature at the
nuclear pore is higher than at the sheet edge. The nuclear pore complex (NPC) likely serves as a
scaffold to stabilize this curvature. In addition, a class of SUN proteins in the INM and KASH
proteins in the ONM interact similar to Climp-63 [25]. Notably, some of the KASH proteins
contain large CC repeats in the cytosolic region [26], analogous to those of kinectin and p180,
suggesting that KASH proteins not only control luminal spacing, but may also smooth the ONM.
Depletion of several ER-localized proteins, including Rab10, Rab18, protrudin, and Lnp, has
been reported to expand ER sheets [10,13,15,16]. If these proteins participate directly in ER
shaping, they either promote tubule formation or negatively regulate ER sheets. Rab10 appears
to promote tubule formation, but how other proteins influence ER morphology is unknown.
Experimentally, we need to determine: (i) whether the effect is direct, as processes such as the
activation of ER stress also result in ER expansion; (ii) whether the change in morphology is
due to the augmentation of new sheets or redistribution of existing sheets; and (iii) whether the
change correlates with increased levels of sheet-forming proteins, such as Climp-63.
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Fusion of ER Membranes
Homotypic fusion, the merging of identical membranes, occurs frequently in the ER, forming a
tubular network and maintaining the continuity of ER membranes. ATLs act as ER fusogens [7,8]
and yeast and plants use the functional orthologs of ATLs, synthetic enhancer of yop1p (Sey1p)
and root hair defective 3 (RHD3), respectively. Purified Drosophila ATL, Sey1p, and RHD3
have membrane fusion activity in vitro, directly supporting their role in ER fusion [8,27,28].
Alteration of ATL or Sey1p/RHD3 profoundly impacts ER morphology. In yeast cells, deletion of
Sey1p and Yop1p results in a conversion of the tubular network into sheets and large areas of
the cortex devoid of ER [7,27]. Mutation or deletion of RHD3 leads to cable-like and less-mobile
ER tubules [29]. Depletion of ATLs generates unbranched ER [7] or, in the case of Drosophila
ATL, fragmented ER [8]. These morphological defects are attributed to a lack of connections
between ER tubules.
Structural studies of human ATL1 and Candida albicans Sey1p have provided significant insight
into the mechanism by which these GTPases fuse membranes [30–33]. ATL and Sey1p/RHD3
contain an N-terminal GTPase and a helical bundle domain, followed by a TMH and C-terminal
tail (CT). GTP binding-induced dimerization of the GTPase promotes membrane tethering, and
GTP hydrolysis induces conformational changes in the helical bundle, forcing the membranes to
merge. In addition, the TMH mediates oligomerization and has sequence-specific functions
and the CT forms an APH, which destabilizes the lipid bilayer during fusion [34].
ER fusion is tightly regulated [35]. Not all tubule meetings proceed to fusion, but drastic loss of
fusion activity causes severe morphological and functional defects. Although regulators of ATL
and Sey1p/RHD3 are yet to be discovered, some insights have come from the intrinsic
properties of these GTPases. Recent studies have revealed that tethered membranes continuously consume GTP but do not necessarily fuse, and GTPases form dimers and hydrolyze GTP
in the same membranes [35]. These apparently futile efforts may be used to self-regulate fusion
dynamics.
ER morphology in Membrane Contact Sites
To maintain cellular homeostasis, organelles are connected by vesicle-mediated trafficking and
membrane contact sites (MCSs) at which two heterologous membranes are closely apposed
(typically within 30 nm) but do not fuse. The ER forms MCSs with multiple membrane systems,
including the plasma membrane (PM), mitochondria, Golgi, endosomes, and lipid droplets (LDs)
(Figure 2). Association of the ER with other organelles generally involves protein–protein
interactions and/or protein–phospholipid interactions. The areas and shapes of such contacts
are dynamic and may correlate with the functional demands of the contact, which include lipid
and calcium exchange, fission, and organelle movement.
ER–PM Contact Sites
The cortical ER, which is tightly associated with the PM, consists of tubules and highly
fenestrated sheets. In yeast, the cortical ER normally covers 20–40% of the PM [3], and
can be separated from the PM only when six tethering proteins, including Ist2, three tricalbins
[Tcb1-3, orthologs of extended synaptotagmin (E-Syt)], Scs2, and Scs22 [orthologs of
VAMP-associated proteins (VAPs)[4_TD$IF]], are simultaneously deleted.
Several pairs of interactions have been identified for ER–PM contacts. The ER-resident calcium
sensor stromal-interacting molecule 1 (STIM1) binds to the PM-localized Ca2+[6_TD$IF] channel Orai1 to
replenish Ca2+ levels in the cytosol when Ca2+stores in the ER are depleted [36]. Binding of
STIM1 with PI(4,5)P2 on the PM enhances contact formation [36]. ER–PM tethering can also be
mediated by numerous lipid transfer proteins. ER-localized E-Syts engage PI(4,5)P2 on the PM
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TICB 1254 No. of Pages 10
ER-PM contacts
PM
STIM1
Orai1
VAP-A/VAP-B
Scs2/Scs22
NIR2
E-syts/
Tcbs
PI(4,5)P2
ORP5 ORP8/
Osh
PI(4)P
PA
SR
Golgi
ER-Golgi contacts
VAP
LD
OSBP
PI(4)P
Sheet
ER
ER-LD contacts
Mitochondria
Seipin/Fld1
Tu
Endosome
ER-endosome contacts
ORP5
NPC1
VAP
ORP1L
Mmm1
STARD3
Rab7
le
ER-mitochondrial
contacts (ERMES)
Mmm12
STARD3NL
Protrudin
bu
IP3R
Mdm10
Mdm34
MCU
PI(3)P
Figure 2. ER-Mediated Organelle Contacts. Schematic illustration of ER-mediated contact sites. The shapes and
areas of ER sites that contact other organelles are dynamic. Both ER tubules and sheets may be involved in responses to
different stimuli. Some of the tether factors and/or effectors that mediate contact formation are listed. In general, ER-resident
proteins are listed on the left, and their corresponding factors are listed on the right. Gray symbols represent proteins that are
not discussed in this review. Abbreviations: ER, endoplasmic reticulum; LD, lipid droplet; SR, sarcoplasmic reticulum.
via their C2 domains [37], and their SMP domain is shown to be a lipid transfer module [38–40].
Such interactions are thought to be sensitive to the levels of cytosolic Ca2+ [41]. The tricalbins
found in yeast are structurally and functionally homologous to E-Syts [42,43]. Following phospholipase C (PLC)-activating stimuli, Nir2 is recruited to ER–PM contact sites, where it transfers
phosphatidic acid (PA) generated in the PM to the ER [44]. The relocation of Nir2 involves VAP in
the ER and PA in the PM. The ER integral membrane proteins ORP5 and ORP8, which bind to PI
(4)P in the PM via their pleckstrin homology (PH) domains, deliver phosphatidyl serine (PS) from
the ER to the PM and counter-transport PI(4)P to the ER for degradation by the ER-resident PI(4)
P phosphatase Sac1 [45].
ER tubules are critical for ER–PM contact sites. Deletion of the tubule-forming protein Rtn4a
dramatically expands ER sheets and reduces store operated calcium entry, which relies on Orai–
STIM interactions [46]. E-Syts are anchored in ER membrane through a typical TMH [37], which
suggests a preference for ER tubules. However, in myocytes, where the SR and PM need to be
continually linked, the contacts are mainly cisternal [47]. Thus, PM interactions are likely initiated
by ER tubules but can evolve into sheets if necessary.
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ER–Mitochondria Contact Sites
The ER attaches to mitochondria at a region known as the mitochondria-associated ER
membrane (MAM). MAMs are important for calcium homeostasis and phospholipid biosynthesis. In yeast, ER–mitochondria contacts are tethered by the ERMES complex[5_TD$IF] [48]. The
molecular basis of the contacts in mammalian cells is not clear. ER tubules, in particular, appear
to be associated with mitochondrial dynamics and function. ER tubules wrap around mitochondria to create mitochondrial constriction sites, where fission factors such as the dynaminlike GTPase Drp1 are recruited and assembled [49]. In yeast cells, such a wrapping interaction
involves the highly conserved Miro GTPase Gem1 in addition to ERMES, and regulates the
distribution of mitochondria and mitochondrial DNA [50].
Coupling of calcium signaling between the two major intracellular Ca2+[5_TD$IF] stores is based on the
crosstalk between the IP3 receptor on the ER side and the mitochondrial Ca2+ uniporter (MCU),
and relies on the physical contact between the two organelles [51]. Interestingly, IP3 receptormediated ER-mitochondria contacts colocalize with ER tubules [52], supporting the tubulebased nature of the contacts.
Mitochondria need phospholipid supplies from the ER, and lipid transfer may benefit from ER–
mitochondria contact sites. Components of the ERMES complex contain SMP domains that
bind to phospholipids [53], and a conserved ER membrane complex facilitates lipid transfer from
the ER to mitochondria [54]. However, contact site-mediated transfer may not be essential in
higher eukaryotes, because in Xenopus egg extracts PS can be transferred from the ER to
mitochondria when the two membranes are no longer tethered [55].
ER–mitochondria contacts also act as sites for autophagosome formation. Recent studies
revealed that autophagosomes form at MAMs in mammalian cells, and impaired ER–mitochondria contacts attenuate autophagosome formation [56]. In yeast, ERMES is dispensable for
nonselective bulk autophagy. However, mitophagy, which involves selective engulfment of
mitochondria by autophagosomes, requires ERMES-established mitochondria–ER contacts
[57]. ERMES functions at the membrane expansion during mitophagy, suggesting that MAMs
may transfer phospholipids to the forming autophagosome.
ER–Golgi Contact Sites
ER–Golgi contacts occur mostly between flattened ER sheets and the trans-most Golgi
cisternae [58]. Different lipid transfer proteins are present at ER–Golgi contacts. Oxysterolbinding proteins (OSBPs) deliver sterol to the Golgi and transfer PI(4)P for degradation by Sac1
[59]. CERT transports ceramide from the ER to the Golgi for sphingomyelin synthesis [60]. The
area of ER–Golgi apposition can be modulated by the levels of lipids and tethering proteins. Golgi
structures are reported to be completely enwrapped by the ER upon 25-hydroxycholesterol
treatment and expression of VAP-A and OSBP [59].
ER–endosome Contact Sites
The ER network forms multiple contacts with endosomes to regulate endosome processes
including fission, maturation, movement, cholesterol transfer, and receptor dephosphorylation [61]. These distinct functions appear to be conferred by different tethering complexes
involving different endosome subpopulations. Tethering of ORP5 with the late endosomal
cholesterol transporter NPC1 facilitates the transfer of cholesterol from late endosomes (LEs)
to the ER [62]. Low cholesterol levels on LEs facilitate the interaction between ORP1L and
VAP-A, which removes the dynein motor subunit p150Glued and associated motors from
Rab7–RILP, resulting in plus-end-directed transport of LEs [63]. The integral ER membrane
protein protrudin interacts with Rab7-GTP and PI(3)P on the endosome membrane to form
ER–LE contact sites, at which the motor protein kinesin 1 is transferred to the motor adaptor
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FYCO1 on LEs, resulting in transport of LEs to the cell periphery [64]. The formation of
ER–endosome contacts creates constrictions of endosomes, which facilitate endosome
fission [65]. Contact sites may provide platforms for recruiting fission machinery or generating
highly curved membrane regions favorable for fission via lipid and Ca2+ transfer. Contact
sites also restrict cargo diffusion, contributing to partitioning endocytic cargo to distinct
endosome domains [65]. Overexpression of Rtn4a causes ER tubules to become less
dynamic and reduces endosome fission [65]. Deletion of RHD3 in plants causes similar
defects [66].
Although most ER–endosome contacts occur with ER tubules, increased levels of the LE
membrane-anchored proteins STARD3 and STARD3NL, which tether the ER by interacting
with VAPs, result in a tight enclosure of endosomes by ER sheets [67]. This impairs the
generation of LE tubules, resulting in less mobile LEs [67]. Therefore, ER dynamics and shape
must be precisely controlled to facilitate endosome dynamics.
ER–LD Contact Sites
Nascent LDs pinch off from ER bulges where neutral lipids accumulate between the phospholipid bilayers. LDs likely form contacts with ER sheets, and these contacts probably link
neural lipid synthesis with LD expansion. The ER-resident transmembrane proteins Fld1 and
seipin are enriched at ER–LD contact sites in yeast and mammalian cells, respectively [68].
Interestingly, LDs also physically connect with the ER via a stalk, which may function as a
conduit for the exchange of lipids and proteins. Factors tethering ER–LD contacts remain
largely unknown.
ER Morphology in Membrane Trafficking
In addition to direct contacts, vesicle-based trafficking is a major pathway for exchanging
materials between membrane systems. Newly synthesized proteins or lipids are packed into
COPII-coated vesicles and leave the ER through ER exit sites (ERESs). Because the curvature of
a vesicle is comparable to that of a tubule cross-section, COPII vesicles are speculated to be
preferentially generated in ER tubules. ERESs are enriched in tubules [17]. Most ER tubules are
peripherally localized, but a significant proportion is clustered near the nucleus due to dyneinbased retrograde movements [69]. COPII trafficking is expected to occur in the perinuclear
region where ER sheets, on which proteins are synthesized, and the Golgi apparatus, where
proteins are processed, both localize. Whether perinuclear tubules harbor the majority of ERES
remains to be assessed.
The connection between membrane trafficking and ER morphology is supported by an auxinsignaling defect occurring with the deletion of RHD3; the major ER fusogen in plants [66]. Auxin
transport relies heavily on coordinated exocytosis and endocytosis. In rhd3 cells, ER mobility and
endosome streaming are drastically affected [66], impairing endocytosis. ER export may also be
inhibited by the lack of ER complexity in rhd3 cells. Similar defects were found in the endocytic
pathway when neurolastin, a brain-specific homolog of ATL, was deleted in a mouse model, and
the consequence was drastically reduced excitatory synapses and spine density [70]. In
mammalian cells, Rtn1 is shown to indirectly influence the function of the Golgi by regulating
Rab1 and Rab43 via TBC1D20 [71]. In addition to the general defects caused by determinants of
ER morphology, individual family members have been implicated in modulating the trafficking of
specific cargos. In Arabidopsis, RTNLB regulates ER export of the FLS2 immune receptor [72];
in mammalian cells, ATL1 mutations are linked to the defective transport of BMP receptor
BMPRII [73] and REEPs affect trafficking of specific G-protein-coupled receptors [74]. Whether
additional cargos will be affected in these cases is not clear, as the cargos available for testing are
limited. Nevertheless, these results suggest that the ER tubular network actively participates in
cargo sorting and provides a platform for vesicle budding.
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ER Morphology in Multicellular Development
Outstanding Questions
ER morphogenesis, especially tubular network formation, appears to be a highly conserved
process in eukaryotes. ER shaping and remodeling are expected to play a fundamental role
during development. Surprisingly, in the basic eukaryotic model, the growth of yeast cells is only
slowed when Yop1p and Rtn1p are deleted, and sey1D cells seem to be normal [7]. The
functions of these proteins could be fulfilled by yet unknown analogous proteins. ER membrane
dynamics have also been suggested to be more critical when cells expand during development
[75]. The cortical ER of cotyledon epidermal cells in rhd3 plants is indistinguishable from that of
wild-type plants at early developmental stages, but as cells grow, the cable-like ER phenotype
starts to appear. In Caenorhabditis elegans, which has much larger cells than yeast, depletion of
both YOP-1 and RET-1 (the homolog of Rtn4a in C. elegans) dramatically reduces the embryo
survival rate [76]. Similarly, deletion of Rtnl1 (the only widely expressed Rtn in Drosophila) causes
ER sheet expansion and defects in distal motor axons [77].
Many proteins have been identified that
are involved in ER morphogenesis,
and their mechanisms of action have
prompted much speculation. How do
tubule-forming proteins assemble into
oligomers? Is Climp-63 really a luminal
spacer? If so, what is the molecular
basis of its activity? How do Kinectin
and p180 contribute to sheet formation? How do ER shaping proteins
coordinate with each other?
When genes that shape the ER have redundancy in the genome, the situation becomes more
complicated. [14_TD$IF]It is thought that deletion or mutation of individual isoforms may not cause a
prominent phenotype. In addition to RHD3, Arabidopsis has two tissue-specific RHD3-like
proteins with ER fusogen activity [28]. However, mutation or deletion of RHD3 alone causes
abnormal cortical ER, cell expansion defects, and short root hairs [28]. Although mammals have
more than 10 RHD-containing proteins, knocking out just Rtn4 is sufficient to convert most ER
tubules into sheets in MEF cells [46].
Several ER-shaping proteins have been implicated in hereditary spastic paraplegia (HSP); a
neurodegenerative disease characterized by axon shortening in corticospinal motor neurons
and progressive spasticity and weakness of the lower limbs [78]: ATL1 (SPG3A), Rtn2 (SPG12),
and REEP1 (SPG31) [79]. A mouse model of REEP1 deletion and a Drosophila model of Rtn2
deletion exhibit characteristics of HSP [77,80]. In general, neurons contain long protrusions, and
the integrity of the ER network is more vulnerable to functional impairment. Whether the defect
lies in ER–PM contacts, membrane trafficking, or organelle mobility is currently unknown.
Concluding Remarks
The network of tubules and sheets in the ER are generated by shared mechanisms using
common protein modules, such as TMHs, APHs, and CCs. Many functions of the ER, including
organelle contacts, rely on its morphological features (see [15_TD$IF]Outstanding [16_TD$IF]Questions). Although ER
sheets serve as a platform for protein synthesis, the tubular ER network provides advantages in
membrane trafficking. Processes such as lipid synthesis, though not discussed here, also
profoundly affect ER shaping [15,65], and are likely affected by ER shape. Recent advancements
in super-resolution microscopy will help uncover new ER shaping and remodeling phenomena.
The precise physiological relevance of ER morphology will also be gradually revealed by model
multicellular organisms.
Acknowledgments
We are grateful to Drs. Isabel Hanson and Alicia Prater for editing the work and to Dr. Sha Sun for help with the figures.
Dr. Hong Zhang was supported by grants from the National Natural Science Foundation of China (NSFC) (31421002,
31561143001, 31225018), the National Basic Research Program of China (2013CB910100), and an International Early
Career Scientist grant from the Howard Hughes Medical Institute. J.H. is supported by the NSFC (31225006), the National
Basic Research Program of China (2012CB910302), and an International Early Career Scientist grant from Howard Hughes
Medical Institute.
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