high-curvature domains of the er are important for the...
TRANSCRIPT
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High-curvature domains of the ER are important for the organization1
of ER exit sites in Saccharomyces cerevisiae2
3
Michiyo Okamoto1+, Kazuo Kurokawa1+*, Kumi Matsuura-Tokita1+, Chieko Saito1,4
Ryogo Hirata1, and Akihiko Nakano1,25
61Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako,7
Saitama 351-0198, Japan82Department of Biological Sciences, Graduate School of Science, The University of9
Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan10
11
+These authors contributed equally to this work.12
*Corresponding author13
Email: [email protected]
15
Running title: Localization of ER exit sites on the ER16
Keywords: ERES, COPII, ER membrane curvature17
Word count: 566718
19Abbreviations used in this paper: COPII, coat protein complex II; ER, endoplasmic20
reticulum; ERES, ER exit site; ERGIC, ER-to-Golgi intermediate compartment; GEF,21
guanine nucleotide exchange factor22
© 2012. Published by The Company of Biologists Ltd.Jo
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JCS online publication date 30 March 2012
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Summary1
Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus2
occurs at specialized regions known as the ER exit sites (ERES). In Saccharomyces3
cerevisiae, ERES show numerous scattered puncta throughout the ER. We examined4
ERES localization within the peripheral ER, finding that ERES localize on high-5
curvature ER domains where curvature-stabilizing protein Rtn1 is present. Δrtn1 Δrtn26
Δyop1 cells have fewer high-curvature ER domains, but ERES accumulate at the7
remaining high-curvature ER domains on the edge of expanded ER sheets. We propose8
that membrane curvature is a key geometric feature for the regulation of ERES9
localization. We also investigated a spatial relationship between ERES and Golgi10
cisternae. Golgi cisternae in S. cerevisiae are unstacked, dispersed, and moving in the11
cytoplasm with cis-cisternae positioned adjacent to ERES, whereas trans-cisternae are12
not. Morphological changes in the ER of Δrtn1 Δrtn2 Δyop1 cells resulted in aberrant13
Golgi structures, including cis-and trans-markers, and exhibited reduced motion at14
ERES between expanded ER sheets and the plasma membrane.15
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Introduction1
The secretory pathway conveys a large number and wide variety of proteins as2
cargo to their final destinations, such as the extracellular space and the plasma3
membrane. The ER is the starting organelle of the pathway, and the Golgi apparatus acts4
as its pivotal sorting station. Recent studies on the budding yeast Saccharomyces5
cerevisiae have revealed that the Golgi apparatus is very dynamic in its structure, and6
the compartments of the Golgi change from cis-cisternae to trans-cisternae over time7
(Losev et al., 2006; Matsuura-Tokita et al., 2006). Though these observations provide8
strong support for the cisternal maturation model, they also raise new questions as to9
how new Golgi are generated and the cargo molecules transported into cis-Golgi (Emr10
et al., 2009; Glick and Nakano, 2009; Nakano and Luini, 2010).11
ER-to-Golgi transport is mediated by coat protein complex II (COPII) vesicles.12
Components responsible for COPII vesicle formation are well conserved between yeast13
and mammals (Kuge et al., 1994; Swaroop et al., 1994; Paccaud et al., 1996; Tang et al.,14
1999; Tang et al., 2000; Weissman et al., 2001; Bhattacharyya and Glick, 2007) and15
include COPII coat protein subunits Sec23, Sec24, Sec13, and Sec31, a small GTPase16
Sar1, and its specific guanine nucleotide exchange factor (GEF) Sec12 (Nakano et al.,17
1988; Nakano and Muramatsu, 1989; Barlowe and Schekman, 1993; Barlowe et al.,18
1994). When activated by Sec12, Sar1-GTP initiates COPII vesicle formation on the ER19
by sequentially recruiting Sec23/24 heterodimers and Sec13/31 heterodimers (Lee et al.,20
2004). Cell-free experiments using synthetic liposomes, proteoliposomes, and a planar21
lipid bilayer have shown that Sec23/24, Sec13/31, and Sar1-GTP are sufficient for22
formation of the COPII vesicle, but multiple rounds of the Sar1 GDP/GTP cycle23
stimulated by Sec12 are pivotal for efficient cargo selection (Matsuoka et al., 1998).24
Another key component of COPII vesicle formation is a peripheral membrane protein,25
Sec16, which directly interacts with Sec23, Sec24, and Sec31 (Espenshade et al., 1995;26
Gimeno et al., 1996; Shaywitz et al., 1997). Sec16 facilitates the recruitment of COPII27
coat proteins on liposomes and stabilizes the coat to prevent premature disassembly28
(Supek et al., 2002). Thus, Sec16 is considered to function as a scaffold for assembling29
COPII coats (Supek et al., 2002; Connerly et al., 2005).30
Previous studies have shown that COPII vesicles are formed at the specialized31
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sites within the ER, termed the ER exit sites (ERES) or the transitional ER. ERES are1
the sites where cargo and COPII coat proteins are concentrated, and are2
morphologically distinct from the surrounding ER (Palade, 1975; Orci et al., 1991;3
Bannykh et al., 1996). Although COPII vesicle formation has been characterized in4
detail, the structure and organization of ERES remain to be elucidated. In mammalian5
cells, COPII components are concentrated in hundreds of punctate structures along the6
ER, enriched at the juxtanuclear region where stacked Golgi exists (Orci et al., 1991;7
Bannykh et al., 1996; Stephens, 2003). Punctate ERES structures are found adjacent to8
the ER-to-Golgi intermediate compartment (ERGIC). Localization of Sec23/24,9
Sec13/31, and Sec16 is limited at ERES, whereas Sar1 is localized throughout the ER10
with some accumulation at ERES (Watson et al., 2006). Sec12 is localized uniformly11
within the entire ER (Weissman et al., 2001). For budding yeast species, the Golgi12
cisternae of Pichia pastoris form a stacked structure, whereas those of S. cerevisiae are13
unstacked and dispersed in the cytoplasm (Orci et al., 1991; Bannykh et al., 1996;14
Rossanese et al., 1999; Bevis et al., 2002; Stephens, 2003). P. pastoris has a small15
number of ERES (two to six per cell), each juxtaposed to the stacked Golgi (Rossanese16
et al., 1999; Bevis et al., 2002). Sec12 and Sar1 accumulate at ERES with COPII coats17
and Sec16 (Soderholm et al., 2004). The structural properties of S. cerevisiae ERES18
were unclear until recently. Live cell imaging demonstrated that S. cerevisiae also has19
organized ERES that consist of numerous punctate structures marked by COPII coat20
proteins (Castillon et al., 2009; Levi et al., 2010; Shindiapina and Barlowe, 2010). Sar121
and Sec12 are reported to localize throughout the ER (Nishikawa and Nakano, 1991;22
Rossanese et al., 1999). The structural differences between the ERES of P. pastoris and23
S. cerevisiae might be reflected in the discrepant features of their Golgi cisternae.24
In mammalian cells and plant cells, punctate ERES localize on the surface of25
cortical ER tubules (Hammond and Glick, 2000; Yang et al., 2005). The ERES of26
budding yeast are found on the nuclear envelope and the peripheral ER (Bevis et al.,27
2002; Shindiapina and Barlowe, 2010), but their precise distribution on the ER28
membrane has not been revealed. Because the ER has an elaborate shape consisting of a29
network of membrane-enclosed tubules and sheets, the distribution of ERES might30
depend on the ER morphology. Although little is known about how the ER structures31
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are generated and maintained, recent studies identified two protein families intimately1
involved in shaping the ER. Reticulons (Rtns) and DP1/Yop1 are curvature-stabilizing2
proteins that localize in the ER tubules and at the edge of ER sheets (De Craene et al.,3
2006; Voeltz et al., 2006; Shibata et al., 2010). Over-expression of Rtns generates4
longer unbranched tubules and fewer sheets, whereas depletion of Rtns results in the5
proliferation of sheet regions at the expense of tubules (Voeltz et al., 2006; Anderson6
and Hetzer, 2008). Atlastin family of proteins, including S. cerevisiae Sey1, have also7
been implicated in organization of tubular network of the ER (Farhan and Hauri, 2009;8
Hu et al., 2009).9
We conducted high-resolution live imaging of ERES in S. cerevisiae for the10
seamless understanding of the ER-to-Golgi system. In the present study we report that11
S. cerevisiae ERES consist of COPII coat proteins and Sec16 and are preferentially12
distributed on high-curvature domains of the ER membrane: ER tubules and the edge of13
ER sheets. Morphological changes of the ER affected the distribution of ERES, but14
ERES still localized on the high-curvature regions of the ER. Correlation analysis15
revealed that cis-Golgi cisternae, but not trans-Golgi cisternae, were juxtaposed to16
ERES. Remarkably, ectopic localization of ERES resulted in the organization of some17
aberrant Golgi cisternae, which were also formed in the vicinity of ERES, but where cis18
and trans cisternal markers acted together. These findings provide new information19
about the relationship between the ERES localization and ER morphology and the20
organization of the ER-to-Golgi system.21
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Results1
Organization of ERES in S. cerevisiae2
ERES is the ER membrane subdomain where COPII vesicles are assembled3
(Orci et al., 1991; Bannykh et al., 1996; Stephens, 2003; Connerly et al., 2005; Watson4
et al., 2006). Recently, Shindiapina and Barlowe (2010) reported that S. cerevisiae5
Sec13-GFP and Sec23-GFP localize to small punctate spots, which were long-lived and6
exhibited restricted motion within the ER, representing ERES in this organism. We7
confirmed that the COPII coat proteins (Sec13, Sec31, and Sec24) co-localized with8
each other and with Sec16, another well established ERES marker. GFP- or mRFP-9
fused COPII coat proteins Sec24, Sec13, and Sec31, and Sec16-GFP all yielded10
punctate patterns of fluorescence. Simultaneous observations of two of these proteins11
indicated their precise co-localization, indicating that COPII coat proteins and Sec1612
accumulate at ERES in S. cerevisiae (Fig. 1A). Sec16 is a peripheral membrane protein13
predicted to be a scaffold for COPII assembly at ERES (Supek et al., 2002; Connerly et14
al., 2005). Consistently, Sec16-GFP puncta appear to be unaffected by the sec12-415
mutation, which induced a couple of coalescence of COPII coat puncta upon shift to the16
restrictive temperature (Fig. 1B). These results suggest that accumulation of the COPII17
coats at ERES depends on Sar1 GTPase activation, but that of Sec16 does not.18
Next, we examined whether Sec12 also accumulate at ERES. Confocal imaging19
near the center of the cell indicated that fluorescent signals of mRFP-Sec12 were20
observed throughout the ER, and scattered patterns of ERES fluorescence were adjacent21
to the Sec12 signals (Fig. 2A, upper panels). Confocal images of the cell periphery22
revealed that Sec13 puncta were positioned around Sec12, but there was little overlap23
between the two (Fig. 2A, lower panels). To confirm this result, two-dimensional24
confocal sections of mRFP-Sec12 and Sec13-GFP fluorescence were reconstructed into25
three-dimensional images, which are shown in Fig.2B. Sec13 puncta were localized on26
the edge of ER membrane labeled by mRFP-Sec12. These results indicate that Sec1227
does not accumulate at S. cerevisiae ERES.28
29
ERES localize on high-curvature domains of the peripheral ER network30
The appearance of mRFP-Sec12 fluorescence was similar to that of the ER31
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sheet structure. We explored whether the localization of ERES correlates with the shape1
of the ER. The ER consists of a network of branching tubules and flat sheets. The2
morphology of the peripheral ER was visualized by GFP targeting to the ER lumen3
(GFP-HDEL). The peripheral ER consisted of both the tubules (Fig. 3A, arrowhead)4
and fenestrated sheets (Fig. 2A, B and 3A, arrows). Rtns and DP1/Yop1 are curvature-5
stabilizing proteins and partition to high-curvature regions of the ER, ER tubules, and6
the edge of ER sheets (De Craene et al., 2006; Voeltz et al., 2006; Shibata et al., 2010).7
Rtn1-GFP signals were found on the tubules and at the rim of the fenestrated sheets8
(Fig. 3B, lower panels). Sec12 was predominantly enriched in the ER sheets because9
Rtn1-GFP fluorescent signals circumscribed those of mRFP-Sec12 (Fig. 3B, lower10
panels). Dual-color observations of Sec13-GFP and Rtn1-mRFP revealed that ERES11
puncta marked by Sec13-GFP always localized at the high-curvature ER domain labeled12
by Rtn1-mRFP fluorescence (Fig. 3C, lower panels). The ERES of budding yeast are13
also found on the nuclear envelope which has much less curvature than the peripheral14
ER (Bevis et al., 2002; Shindiapina and Barlowe, 2010). Dual color observation of15
mRFP-Sec12 and Rtn1-GFP showed that high-curvature domains of ER membrane16
labeled by Rtn1-GFP distributed not only at the cell periphery but also on the nuclear17
envelope (Fig.3B, upper panels). We also found that these punctate signals of Rtn1-GFP18
on the nuclear envelope localized at the bases of the ER tubules and central cisternal ER19
(Fig.3B, upper panels, arrowhead) (West et al., 2011). Furthermore, Sec13 colocalized20
with Rtn1 on the nuclear envelope (Fig.3C, lower panels). These results indicate that the21
ERES are preferentially distributed on the high-curvature domains of the ER: ER22
tubules and the edge of ER sheets. We also examined ERES localization in relation to23
the surface geometry of the ER membrane. Dual-color confocal images of Sec13-GFP24
and mRFP-Sec12 were reconstructed into three-dimensional data and visualized by the25
isosurface mode of Volocity software (Fig.3D). Their quantitative analysis showed that26
ERES preferentially faced the saddle-shape surfaces of the high-curvature ER27
membrane, saddle shape meaning convex (positive curvature) toward the edge and28
concave (negative curvature) along the edge (Fig. 3 E) (Zimmerberg and Kozlov, 2005).29
Collectively, our results suggest that the distribution of ERES is governed by the30
geometric features of the ER.31
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1
Disruption of the peripheral ER network changes ERES distribution2
The restricted localization of ERES on the high-curvature domains of the ER3
prompted us to examine whether morphological changes of the ER influence the4
distribution of ERES. We examined ERES distribution in Δrtn1 Δrtn2 Δyop1 cells and5
Δsey1 Δyop1 cells (Voeltz et al., 2006; Hu et al., 2009). Both mutants exhibit normal6
proliferation and secretory properties, but they are defective in the formation of ER7
tubules and accumulate longer peripheral ER sheets (Voeltz et al., 2006). Confocal8
imaging of mRFP-Sec12 or HDEL-GFP at the cell periphery of these mutants clearly9
showed that they have continuous unfenestrated peripheral ER sheets lacking a tubular10
network (Fig. 4A). The number of ERES in these mutant cells was almost the same as11
the number in wild-type cells (Fig. 4B). Dual-color observations of Sec13p-GFP and12
mRFP-Sec12 at the cell periphery showed that ERES in these mutant cells clustered, in13
contrast to the scattered pattern in wild-type cells (Fig. 2A); however, they still14
associated with the remaining edge of the enlarged ER sheets and avoided the flat15
surface of these sheets (Fig. 4A). These data indicate that ERES distribution is affected16
by morphological changes in the ER, and that ERES localization is restricted to the17
high-curvature domains of the ER.18
19
cis-Golgi cisternae are in the vicinity of ERES20
According to the cisternal maturation model of Golgi, ERES are birth places21
for new Golgi cisternae (Glick and Malhotra, 1998). Consistent with this idea, ERES in22
cells with stacked Golgi cisternae have been shown to be positioned adjacent to the cis-23
side of the Golgi apparatus, implying that ERES are the origin of the Golgi (Rossanese24
et al., 1999; Kondylis and Rabouille, 2003; Yang et al., 2005). Golgi cisternae are not25
stacked in S. cerevisiae. The cisternae are scattered throughout the cytoplasm while they26
mature. Thus in this organism, the positional relationship between the Golgi and ERES27
has been less obvious (Rossanese et al., 1999). However, we thought that cis-Golgi28
could still localize in closer vicinity to ERES than trans-Golgi in S. cerevisiae. We29
observed ERES, cis-Golgi, and trans-Golgi proteins by dual-color fluorescence confocal30
microscopy (Fig. 5). The cis-Golgi markers Sed5 and Rer1 sometimes almost31
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overlapped with Sec13. Overlapping Sec7 and Sec13 signals were rarely observed. To1
quantify the extent of spatial proximity, we calculated Pearson’s correlation coefficients2
between two fluorescent signals of each combination of proteins in a single confocal3
plane, confirming that cis-cisternae are more closely associated with ERES than trans-4
cisternae (Fig. 5). This finding demonstrates that S. cerevisiae also exhibits a spatial5
relationship between ERES and cis-Golgi as described in other cells with stacked Golgi6
cisternae.7
8
Disruption of the peripheral ER network alters the dynamics of Golgi cisternae9
Golgi and ERES show different dynamics in S. cerevisiae. Golgi cisternae10
are mobile, mature progressively, and dissipate within minutes (Losev et al., 2006;11
Matsuura-Tokita et al., 2006), whereas ERES are reported immobile and stable12
(Shindiapina and Barlowe, 2010). In addition, ERES outnumber Golgi cisternae13
(Rossanese et al., 1999). We were interested in how these compartments maintain their14
spatial proximity and conducted dual-color time-lapse imaging of ERES and cis-Golgi.15
In wild-type cells, cis-Golgi labeled with mRFP-Sed5 frequently localized in the16
vicinity of ERES without being retained at the ERES (Fig. 6A, upper panels). This17
result suggested that new cis-Golgi cisternae are generated at ERES de novo or pre-18
existing cis-Golgi approaches dynamically to ERES.19
Next, we examined whether morphological alteration of the ER affects the20
dynamic features of S. cerevisiae Golgi. In Δrtn1 Δrtn2 Δyop1 cells, we found that some21
cis-Golgi remained near the aligned ERES for a much longer time (Fig. 6A,C). In wild-22
type cells, the peripheral ER and the plasma membrane are closely apposed in a way23
that ribosomes are excluded from the plasma membrane face of the peripheral ER (West24
et al., 2011). In Δrtn1 Δrtn2 Δyop1 cells, some interstices were found between the25
expanded peripheral ER and plasma membrane, where ERES were ectopically located26
on the plasma membrane side of the interstices (Fig. 6B, arrowhead). Dual-color time-27
lapse images showed that cis-Golgi localized on the plasma membrane side of the ER28
were constantly positioned in the vicinity of ERES, whereas cis-Golgi on the29
cytoplasmic side of the ER were not (Fig. 6C). Remarkably, the spatial relationship30
between cis- and trans-Golgi cisternae also changed. We found some cis- and trans-31
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Golgi cisternae exhibited reduced motion and were localized adjacent to each other with1
some overlap, which were seldom observed in wild-type cells (Fig. 6D) (Losev et al.,2
2006; Matsuura-Tokita et al., 2006). These findings suggest that the ectopic localization3
of ERES caused by the morphological change in the ER influenced the dynamic4
behavior of the Golgi apparatus.5
Because the co-localization of cis- and trans-cisternae might indicate6
structural changes, we decided to examine the morphology of the Golgi apparatus in7
Δrtn1 Δrtn2 Δyop1 cells and Δsey1 Δyop1 cells using electron microscopy. As shown by8
fluorescence microscopy, the ER membrane formed longer continuous structures in9
these mutant cells (Fig. 7B,C). Strikingly, aberrant membrane structures (rings or10
concentric circles) were often found in the interstice between the ER and the plasma11
membrane (Fig. 7B-E). This structure also stained by the PATAg method that detects12
polysaccharides, indicating that this structure derive from the Golgi apparatus (Fig. 7F13
and G). To specify whether this structure is the Golgi cisternae stably associated with14
ERES in fluorescence microscopy, we examined immuno-gold staining for cis- and15
trans- Golgi marker proteins, Sed5 and Sec7 (Fig. 7H). The staining pattern indicated16
that these membrane structures have a high oligosaccharide content and contain both17
Sec7 (10-nm gold particles, white arrow) and Sed5 (6-nm gold particles, black arrow).18
These results indicate that ring or concentric-circular membranous structures are19
deformed Golgi apparatus including cis- and trans-cisternae.20
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Discussion1
In this study, we demonstrated that organized S. cerevisiae ERES structures2
marked by COPII coat proteins co-localize with Sec16 but not Sec12. A recent study3
documented that sec12-4 and sec16-2 mutations alter the localization of GFP-tagged4
COPII coat proteins (Castillon et al., 2009; Shindiapina and Barlowe, 2010), indicating5
that Sar1-GTP and Sec16 have roles in the maintenance of ERES. Here, we show that,6
in the sec12-4 mutant at a restrictive temperature, COPII coats are clustered into a large7
structure, but Sec16-GFP remains, exhibiting punctate structures. These large clusters of8
COPII coats labeled by Sec13-mRFP puncta still contain Sec16-GFP (Sup Fig1). These9
results suggest that Sec16 acts early in the Sar1 GTPase cycle and is a primary10
determinant of ERES formation in S. cerevisiae (Supek et al., 2002; Connerly et al.,11
2005; Watson et al., 2006; Bhattacharyya and Glick, 2007; Ivan et al., 2008; Hughes et12
al., 2009). On the other hand, we found that Sec12 is prominently distributed in the ER13
sheets and does not accumulate at ERES. In P. pastoris, Sec12 localizes at ERES, but S.14
cerevisiae-P. pastoris chimeric Sec12 is distributed throughout the ER and does not15
perturb the localization of ERES and Golgi components (Soderholm et al., 2004).16
Therefore, the accumulation of Sec12 at ERES is not required for ERES formation.17
Previous observations indicated that ERES localize on the surface of ER18
tubules in mammalian cells and plant cells (Hammond and Glick, 2000; Yang et al.,19
2005). As expected, the preferential partition of ERES into the high-curvature domains20
of ER became more apparent in cells lacking Rtns and Yop1 or Sey1 and Yop1. We21
found that ERES were clustered at the remaining high-curvature domains at the edge of22
the continuous unfenestrated ER sheets even in these mutants. Therefore, these results23
suggest that high-curvature domains of the ER membrane are required for the24
localization of ERES.25
ERES were first identified morphologically as vesiculating ER regions devoid26
of bound ribosomes (Palade, 1975). Thus, the question becomes whether high-curvature27
domains of the ER represent the functional ER domains. Recent confocal fluorescence28
microscopic analysis showed that the components of the translocation complex are29
enriched in the ER sheets relative to the ER tubules, which indicates that sheets may30
have more bound ribosomes per surface area than tubules (Shibata et al., 2010). Voeltz31
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and colleagues directly measured the ribosome density of the ER domains in S.1
cerevisiae, finding that the cytoplasmic side of the peripheral ER sheets has a high2
ribosome density, but the plasma membrane side rarely has ribosomes (West et al.,3
2011), indicating that the edge of the peripheral ER sheets is a boundary between4
domains with high and low ribosome density. Voeltz and colleagues also reported that5
ER tubules are low ribosome density domains (West et al., 2011). Therefore, our6
findings validate that the distribution of ERES at the high-curvature domains, the edge7
of sheets and tubules, is closely related to the functional compartmentalization of the8
ER.9
In vitro experiments will be required to determine the mechanism of ERES10
formation restricted at the high-curvature domains of the ER surface, whether different11
membrane curvature affects the efficiency of COPII assembly. However, the structural12
information for ERES components might provide a clue about this mechanism. The13
structure of the Sar1/Sec23/24/cargo pre-budding complex is a concave surface14
associated with its membrane-orientated face (Bi et al., 2002). Balch and colleagues15
suggested that Sec23-24 first forms an oligomer, coalescing as minimal tetramer16
clusters of the Sar1/Sec23/24/cargo pre-budding complex to define a site for Sec13-3117
recruitment (Stagg et al., 2008). Thus, the concave surface of these tetramer clusters18
might recognize the high-curvature domains of the ER. It should be noted, however,19
that vesicle budding requires not only positive curvature but also negative curvature to20
be constricted at the neck. Our observations may have some interesting implications21
here, because ERES appear to prefer saddle-like structures which contain both positive22
and negative curvatures. In mammalian cells, COPII vesicles found different ER23
cisternae were closely juxtaposed and protrude into a central region containing a24
collection of vesicles and tubular elements comprising vesicular tubular clusters,25
suggesting that the bases of these ER tubules surrounding the vesicular tubular clusters26
might have negative curvatures (Bannykh et al., 1996). Recently, ERES labeled by27
Sec16A has been reported to localize to concave cup-shaped structures of the ER28
membrane which have negative curvatures (Budnik and Stephens, 2009; Hughes et al.,29
2009). These structures might have both positive and negative curvatures as well,30
because most of ERES in mammalian cells also localize on the surface of ER tubules.31
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Saddle-like ER membrane may be rich in a variety of lipid components including cone-1
shaped and reverse-cone-shaped lipids. Dual-color imaging and correlation analysis2
indicated that cis-Golgi cisternae are not randomly dispersed, but present in the vicinity3
of ERES, whereas no such correlation was found for trans-cisternae. Recent work4
showed that the enlarged S. cerevisiae ERES are formed as the result of slowed ER5
export and often seen in close proximity to cis-Golgi cisternae (Levi et al., 2010).6
Therefore, our findings and previous report provide support for ERES as originating the7
Golgi.8
An unexpected result of our work was the association of cis- and trans-Golgi9
cisternae after morphological alteration of the ER. In mammalian cells, Golgi stack10
formations involve GRASP family proteins, which localize to cis- and medial-trans11
cisternae (Seemann et al., 2000; Xiang and Wang, 2010). Biochemical studies have12
shown that GRASP65 forms stable homodimers, and homodimers residing on adjacent13
Golgi membranes form oligomers. These trans-oligomers are capable of holding the14
cisternal membranes together in stacks (Wang et al., 2003; Wang et al., 2005). S.15
cerevisiae has Grh1, a homolog of GRASP, but lacks Golgi stacks in wild-type cells and16
Δgrh1cells (Levi et al., 2010). However, in Δrtn1 Δrtn2 Δyop1 cells, deformed Golgi17
structures including cis- and trans-cisternae were generated in the space between the18
plasma membrane and the expanded ER sheets. These cisternae remain in the vicinity of19
ERES and show reduced motion. Therefore, early and late Golgi cisternae may exhibit20
associated structures by repressing their motion at ERES because they mature21
progressively. Important questions remain, including whether the dynamics of Golgi22
cisternae determine their own structures, which will require further high-resolution23
imaging.24
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Materials and methods1
2
Yeast strains and culture conditions3
The S. cerevisiae strains and plasmids used in this study are listed in Table I. Cells4
were grown in MCD medium [0.67% yeast nitrogen base without amino acids (Difco5
Laboratories Inc.), 0.5% casamino acids (Difco Laboratories Inc.), and 2% glucose]6
with appropriate supplements. For live imaging, cells were grown at 23°C to the early7
logarithmic phase.8
9
GFP and mRFP constructs10
Strains expressing fluorescent protein-tagged Sec13, Sec16, Sec23 or Sec31 were11
constructed as described in the yeast GFP database at the University of California, San12
Francisco (Huh et al., 2003). GFP-Sed5 and mRFP-Sec12 were expressed under the13
control of the TDH3 promoter on the low-copy plasmid pRS316 or pRS314 (Sato et al.,14
2001). Sec7-mRFP and Sec7-3HA were expressed similarly except that the ADH115
promoter was used instead of the TDH3 promoter.16
17
Fluorescence microscopy18
Throughout this study, we used the super-resolution confocal live imaging19
microscope (SCLIM), which we developed by combining a high-speed and high-signal-20
to noise-ratio spinning-disk confocal scanner (Yokogawa Electric, Japan), cooled image21
intensifies (Hamamatsu Photonics, Japan), and high sensitive HARP cameras (NHK and22
Hitachi Kokusai Electric, Japan) or EM-CCD cameras (Hamamatsu Photonics, Japan)23
(Matsuura-Tokita et al., 2006). High space resolution was achieved by oversampling24
and deconvolution (Nakano and Luini, 2010). Three dimensional images were25
reconstructed and deconvoluted by the parameters optimized for the spinning-disk26
confocal scanner using Volocity software (Perkin Elmer, MA). Temperature-sensitive27
mutants were observed using a thermocontrol stage (Tokai Hit, Japan) at either a28
permissive or restrictive temperature. Pearson’s correlation coefficients were calculated29
for two fluorescent signals to estimate spatial proximity using Volocity software (Perkin30
Elmer, MA).31
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1
2
Electron microscopy3
Cells were rapidly frozen in a high-pressure freezer (HPM010, Bal-Tec Inc.,4
Germany) and transferred to 2% OsO4 in anhydrous acetone pre-cooled in liquid5
nitrogen. Samples were kept at –80°C for 7 days, –20°C for 2 h, 4°C for 2 h, then at6
room temperature for 2 h. After a wash with anhydrous acetone, the samples were7
embedded in Spurr’s resin (Nisshin EM, Japan). Ultrathin sections were cut, stained8
with uranyl acetate and lead citrate, and observed under a transmission electron9
microscope (JEM1200EX, JEOL, Japan). For oligosaccharide staining, two sections10
were collected independently, one on a copper grid for conventional structural11
observation and the other on a gold grid for oligosaccharide staining by periodic acid-12
thiocarbohydrazide-silver protein (PATAg) (Thiery and Bader, 1967).13
For immunoelectron microscopy, samples were fixed as described above and14
transferred to 0.01% OsO4 in anhydrous acetone. The samples were kept at –80°C for 715
days, –20°C for 2 h, and 4°C for 2 h. After washing with anhydrous ethanol, the16
samples were embedded in LR-white resin. Polymerization was carried out at –20°C17
using a UV polymerizer (TUV-200, Dosaka-EM, Japan). Ultrathin sections were cut,18
immunolabeled, and stained with uranyl acetate. For immunodetection, rabbit anti-GFP19
polyclonal antibody (1:50; Invitrogen, CA) and 6-nm gold goat anti-rabbit conjugate20
(1:50 dilution; Jackson ImmunoResearch, CA) were used as primary and secondary21
antibodies to detect GFP-Sed5, and mouse anti-HA monoclonal antibody 12CA5 (1:25,22
16 µg/ml, Roche, Switzerland) and 10-nm gold goat anti-mouse conjugate (1:5023
dilution; Zymed, CA) were used as primary and secondary antibodies to detect Sec7-24
3HA.25
26
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Acknowledgements1
This work was supported by a Grant-in-Aid for Specially Promoted Research from the2
Ministry of Education, Culture, Sports, Science and Technology of Japan and by the3
funds from the Bioarchitect, the Extreme Photonics, and the Cellular Systems Biology4
Projects of RIKEN. We thank T. A. Rapoport and Y. Shibata of Harvard University for5
the yeast reticulon mutant strains and G. K. Voeltz of the University of Colorado at6
Boulder for exchange of information prior to publication. We also thank A. Hirata of7
the University of Tokyo for EM technical suggestions and Y. Suda, K. Fukaya, Y.8
Sugisawa, R. Kiuchi, and Y. Zenke of the Nakano Laboratory for assistance and helpful9
suggestions.10
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Figure legends1
2
Figure 1. Localization of COPII coat proteins and Sec16 in wild-type and sec12-43
cells. (A) Dual-color images of wild-type cells expressing Sec24-GFP and Sec13-mRFP,4
Sec24-GFP and Sec16-mRFP, and Sec16-GFP and Sec13-mRFP. GFP and mRFP co-5
localized on numerous puncta, outlining the nuclear envelope and the peripheral ER. (B)6
The sec12-4 cells expressing Sec24-GFP, Sec13-GFP or Sec16-GFP were observed at a7
permissive temperature (23°C), and after incubation for 30-60 min at a restrictive8
temperature (37°C). Fluorescence of COPII coat proteins, Sec24 and Sec13 was9
dispersed in the cytoplasm, and a few large structures were seen. Sec16-GFP puncta did10
not change at 37°C. The sec12-4 cells expressing GFP-Rer1 were used as a control to11
check the inhibition of transport. Scale bars = 5 µm in (A and B).12
13
Figure 2. Sec12 does not accumulate at S. cerevisiae ERES Dual-color confocal14
images of wild-type cells marked with Sec13-GFP and mRFP-Sec12. (A) Confocal15
images near the center (upper panels) or at the periphery (lower panels) . Arrows in the16
lower panels indicate fenestration of the peripheral ER marked with mRFP-Sec12. (B)17
Three dimensional images were reconstructed and deconvolved by the parameters18
optimized for the spinning-disk confocal scanner. Magnified images of a boxed area19
observed from an arrow direction showed that mRFP-Sec12 did not overlap with ERES20
labeled by Sec13-GFP. Scale bars = 2.5 µm in (A and B).21
22
Figure 3. ERES distribution at the high-curvature domains of the peripheral ER.23
(A) Confocal images of wild-type cells expressing HDEL-GFP near the center of the24
cell (left panel) or at the periphery of the cell (middle panel). Three dimensional image25
of peripheral region is shown in the right panel. The peripheral ER network consisted of26
fenestrated sheet (arrow) and tubule (arrowhead) structures. (B) Dual-color confocal27
images near the center (upper panels) or at the periphery (lower panels) of wild-type28
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cells marked with Rtn1-GFP and mRFP-Sec12. These images were deconvolved by the1
parameters optimized for the spinning-disk confocal scanner. Arrowhead indicates that2
Rtn1-GFP puncta localized at the base of the ER tubules and central cisternal ER3
protruded from the nuclear envelope (upper panels). Rtn1-GFP signals encircled those4
of mRFP-Sec12 (lower panels). (C) Dual-color confocal images near the center (upper5
panels) or at the periphery (lower panels) of wild type cells expressing Sec13-GFP and6
Rtn1-mRFP. These images were deconvolved by the parameters optimized for the7
spinning-disk confocal scanner. The punctate signals of Sec13-GFP were localized at the8
Rtn1-mRFP dots in the nuclear envelope (upper panels) and at the edge of the peripheral ER9
sheets and on the peripheral ER tubules which were labeled by Rtn1-mRFP (lower10
panels). (D) Dual-color three dimensional isosurface images of Sec13-GFP and mRFP-11
Sec12 show ERES localization on the surface of the peripheral ER sheets. Arrows12
indicate ERES localized on the saddle-shaped surface of the sheet edge. The arrowhead13
indicates ERES localized on the expanded surface of the sheet edge. (E) Relative14
percentage of ERES localization for each surface. Scale bars = 2.5 µm in (A-D).15
16
Figure 4. ERES distribution is affected by the loss of high-curvature domains of17
the ER. (A) Dual-color confocal images of the periphery Δrtn1 Δrtn2 Δyop1 cells18
expressing Sec13-GFP and mRFP-Sec12 and Δsey1 Δyop1 cells expressing HDEL-GFP19
and Sec13-mCherry. Sec13-GFP and Sec13-mCherry signals clustered along the edge of20
expanded ER sheets. (B) The average numbers of ERES in wild-type, Δrtn1 Δrtn221
Δyop1, and Δsey1 Δyop1 cells. Scale bar = 2.5 µm in (A and B).22
23
Figure 5. cis-Golgi proteins are located in the proximity of ERES. Wild-type cells24
were marked with Sec13-GFP and cis-Golgi marker mRFP-Sed5, Sec13-mRFP and cis-25
Golgi marker GFP-Rer1, and Sec13-GFP and trans-Golgi marker Sec7-mRFP. Pearson’s26
correlation coefficients between the green and red fluorescent signals were calculated.27
Scale bar = 2.5 µm.28
29
Figure 6. Morphological changes in the ER affect Golgi cisternae dynamics. (A)30
Time-lapse observation of wild-type and Δrtn1 Δrtn2 Δyop1 cells expressing Sec13-31
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GFP and mRFP-Sed5. Images were obtained by focusing on the periphery of the cell.1
(B) Dual-color confocal images of the center of Δrtn1 Δrtn2 Δyop1 cells expressing2
Sec13-GFP and mRFP-Sec12. The arrowhead indicates ERES localized between the3
plasma membrane and peripheral ER sheets. (C) Comparison of the dynamics of cis-4
Golgi cisternae localizing on the cytoplasmic (Cvt) or plasma membrane (PM) side of5
ERES in Δrtn1 Δrtn2 Δyop1 cells. Time-lapse images of Sec13-GFP and mRFP-Sed56
are shown. (D) Time-lapse observation of wild-type and Δrtn1 Δrtn2 Δyop1 cells7
expressing GFP-Sed5 and Sec7-mRFP. Images were obtained by focusing on the8
periphery of the cell. Scale bars = 2.5 µm in (A-D).9
10
Figure 7. Morphological changes in the ER affect Golgi structures. (A-C)11
Ultrastructures of wild-type (A), ∆rtn1 ∆rtn2 ∆yop1 (B), and ∆sey1 ∆yop1 (C) cells as12
visualized by electron microscopy. (D, E) Magnified images of boxed areas in (B) and13
(C). Ring or concentric circle structures were often found between the ER and the14
plasma membrane. (F, G) Higher magnification of concentric circle structures in ∆rtn115
∆rtn2 ∆yop1 cells. One of two serial sections was stained with uranium (F), and the16
other was stained for carbohydrate using the PATAg technique (G). Some small black17
dots on and around the ring structure are PATA-negative stains. Oligosaccharide-18
positive signals were detected. (H) Immuno-gold labeling of the thin-section electron19
microscope images of the concentric circle structure aggregates in Δrtn1 Δrtn2 Δyop120
cells harboring GFP-SED5/SEC7-3HA plasmid. Anti-GFP and anti-HA antibodies were21
used to attach the gold. The anti-GFP antibody was conjugated with 6-nm colloidal gold22
particles and the anti-HA antibody with 10-nm gold particles. Both GFP-Sed5 (black23
arrowhead) and Sec7-3HA (white arrowhead) were detected in the concentric circle24
membrane structure.25
Supplementary figure 126The sec12-4 cells expressing Sec16-GFP and Sec13-mRFP were observed at a27permissive temperature (23°C), and after incubation for 30-60 min at a restrictive28temperature (37°C). Fluorescence of COPII coat proteins, Sec13 was dispersed in the29cytoplasm, and a few large structures were seen. These coalescent structures of Sec1330colocalized with Sec16-GFP puncta which did not change at 37°C.31
32
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A Sec24-GFP Sec13-mRFP Merged
Sec24-GFP Sec16-mRFP Merged
Sec16-GFP Sec13-mRFP Merged
B23°C
37°C
sec12-4Sec24-GFP Sec13-GFP Sec16-GFP Rer1-GFP
Fig. 1
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mRFPGFP Merged
Fig. 2
A
B
GFP MergedmRFP
perip
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3D reconstruction
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A HDEL-GFP
B C
perip
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riphe
ry
Rtn1-GFP MergedmRFP-Sec12
cent
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perip
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Merged
3D reconstruction2D
Sec13-GFP Rtn1-mRFP
cent
er
Sec13-GFP+mRFP-Sec12DMagnified images
E
Loca
lizat
ion
(%)
expanded saddle-shaped
Sheet 0
20
40
60
Fig.3
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Sec13-GFP mRFP-Sec12 Merged
∆rtn1∆rtn2∆yop1
Sec13-mCherry HDEL-GFP Merged
∆sey1∆yop1
Fig.4
A
B
0
20
40
60
80
100
∆rtn1∆rtn2∆yop1
∆sey1∆yop1WT
Ave
rage
num
ber
of E
RE
S
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Fig. 5
Pearson's correlation
Sec13-GFP+ Sec7-mRFP
Sec13-GFP+ mRFP-Sed5
Sec13-mRFP+ GFP-Rer1
0.115 ± 0.076 (n=93)0.412 ± 0.107 (n=91) 0.253 ± 0.079 (n=90)
Jour
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0 8 16 24 4832 40 56 (s) WTSec13-GFP+mRFP-Sed5
∆rtn1∆rtn2∆yop1
A
B
∆rtn1∆rtn2∆yop1
Sec13-GFP mRFP-Sec12 Merged
0 8 16 24 4832 40 56 (s)
0 5 10 15 20 25 403530 45 50 55 60 (s)
0 5 10 15 20 25 403530 45 50 55 60 (s)
Cyt
PM
∆rtn1∆rtn2∆yop1
CSec13-GFP+mRFP-Sed5
DGFP-Sed5+Sec7-mRFPWT
∆rtn1∆rtn2∆yop1
0 8 16 24 4832 40 56 (s)
0 8 16 24 4832 40 56 (s)
Fig.6
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200 nm
A B
F
100 nm
H
500 nm 500 nm
200 nm500 nm
C D E
Fig. 7
G
200 nm
ER
PM
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Table I
Strains used in this study
Strain Genotype Source
YPH499 MATa ura3-52 lys2-801ade2-101trp1-∆63_his3-∆200
leu2-∆1
Sikorski and Hieter,
1989
BY4741 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 Invitrogen
SMY80 MATa sec12-4 ade2 trp1 ura3 leu2 his3 lys2 Laboratory strain
NDY257 BY4741 rtn1::kanMX4 rtn2::kanMX4 yop1::kanMX Voeltz et al., 2006
YMO127 BY4741 sey1::kanMX6 yop1::kanMX6 This study
KMY101-31R YPH499 SEC13-GFP::His3MX6 SEC31-
mRFP::TRP1
This study
KMY102-31R YPH499 SEC16-GFP::His3MX6 SEC31-mRFP::TRP1 This study
KMY103-13R YPH499 SEC24-GFP::His3MX6 SEC13-mRFP::TRP1 This study
KMY104 YPH499 RTN1-GFP::His3MX6 This study
KMY105 YPH499 SEC13-mRFP::TRP1 This study
KMY101-RtR YPH499 SEC13-GFP::TRP1_RTN1-mRFP::TRP1 This study
KMY111 SMY80 SEC13-GFP::TRP1 This study
KMY112 SMY80 SEC16-GFP::TRP1 This study
KMY113 SMY80 SEC31-GFP::TRP1 This study
KMY114 SMY80 SEC24-GFP::TRP1 This study
KMY115 NDY257 SEC13-GFP::TRP1 This study
YMO129 YMO127 SEC13-mCherry::natNT2 This study
YMO168 SMY80 SEC16-GFP::TRP1 SEC31-
mRFP::KanMX6
This study
Plasmids used in this study
Plasmid Description Source
SEC7-mRFP pRS316 (CEN URA3)-PADH1-SEC7-
mRFP
Matsuura-Tokita et
al., 2006
GFP-RER1 pRS316-PTDH3-GFP-RER1 Matsuura-Tokita et
al., 2006
GFP-SED5 pRS316-PTDH3-GFP-SED5 This study
mRFP-SEC12 pRS314 (CEN TRP1)-PTDH3-mRFP-
SEC12
This study
mRFP-SED5 pRS316-PTDH3-mRFP-SED5 Matsuura-Tokita et
al., 2006
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nal o
f Cel
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SAR1-GFP pRS316-PADH1-SAR1-GFP This study
GFP-SED5/SEC7-mRFP pRS316-PTDH3-GFP-SED5-PADH1-SEC7-mRFP This study
GFP-SED5/SEC7-3HA pRS316-PTDH3-GFP-SED5-PADH1-SEC7-3HA This study
pMO13 YCp50 (CEN URA3)-PTDH3-GFP-HDEL Okamoto et al.,
2006
pFA6a-GFP(S65T)-TRP1 for C-terminal GFP-tagging, TRP1 Longtine et al.,
1998
pFA6a-GFP(S65T)-
His3MX6
for C-terminal GFP-tagging, His3MX6 Longtine et al.,
1998
pFA6a-mRFP-TRP1 for C-terminal mRFP-tagging, TRP1 This study
pFA6a-mCherry-natNT2 for C-terminal mCherry-tagging, natNT2 This study
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Figure S1
37°C
23°C
Sec13-mRFP Sec16-GFP Mergedsec12-4
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