Organelle association visualized by three-dimensional
ultrastructural imaging of the yeast cell
Andreas Perktold1, Bernd Zechmann1, Günther Daum2 & Günther Zellnig1
1
Institut für Pflanzenwissenschaften, Karl-Franzens-Universität Graz, Austria; and 2Institut für Biochemie, Technische Universität Graz, Austria
Correspondence: Dr Günther Zellnig,
Institut für Pflanzenwissenschaften,
Universität Graz, Schubertstr.51, A-8010
Graz, Austria. Tel.: 143 316 3805631;
fax: 143 316 3809880; e-mail:
[email protected]
Received 10 November 2006; revised 30
January 2007; accepted 6 February 2007.
First published online 10 April 2007.
DOI:10.1111/j.1567-1364.2007.00226.x
Editor: André Goffeau
Keywords
Saccharomyces cerevisiae ; three-dimensional
reconstruction; cell ultrastructure; organelle
association; mitochondria; endoplasmic
reticulum.
Abstract
This study was aimed at a better understanding of organelle organization in the
yeast Saccharomyces cerevisiae with special emphasis on the interaction and
physical association of organelles. For this purpose, a computer aided method
was employed to generate three-dimensional ultrastructural reconstructions of
chemically and cryofixed yeast cells. This approach showed at a high level of
resolution that yeast cells were densely packed with organelles that had a strong
tendency to associate at a distance of o 30 nm. The methods employed here also
allowed us to measure the total surface area and volume of organelles, the number
of associations between organelles, and the ratio of associations between organelles
per surface area. In general, the degree of organelle associations was found to be
much higher in chemically fixed cells than in cryofixed cells, with endoplasmic
reticulum/plasma membrane, endoplasmic reticulum/mitochondria and lipid
particles/nuclei being the most prominent pairs of associated fractions. In
cryofixed cells, similar preferences for organelle association were seen, although at
lower frequency. The occurrence of specific organelle associations is believed to be
important for intracellular translocation and communication. Membrane contact
as a possible means of interorganelle transport of cellular components, especially of
lipids, is discussed.
Introduction
Organelles of all eukaryotic cells communicate with each
other for various reasons. Firstly, transfer of metabolites
between compartments is required to maintain the cellular
balance of pathways distributed among different organelles.
As an example, steps of the glyoxylate cycle are distributed
between mitochondria and peroxisomes. Secondly, transfer
of components is needed for the exchange of information
between subcellular fractions. Prominent examples of this
process are the transport of mRNA from the nucleus to the
ribosomes and various routes of signal transduction. Finally,
interorganelle transfer of components such as proteins or
lipids is a prerequisite to maintain structure and function
of subcellular compartments. As most organelles are not
autonomous with respect to their molecular equipment, the
aspect of organelle communication deserves our special
attention.
Components can migrate between organelles by diffusion, with the aid of helper proteins, by vesicle flux or
through membrane contact. Small molecules such as metabolites or ions are mainly transported as monomers with or
FEMS Yeast Res 7 (2007) 629–638
without protein catalysis, whereas larger molecules, e.g.
polypeptides, migrate in most cases through receptor
mediated processes or by vesicle flux. The most prominent
example with that respect is protein secretion, which requires a complex machinery to translocate proteins from the
interior of the cell to the cell periphery. Intracellular
translocation of lipophilic components is a specific task,
because for biophysical reasons and as a result of their
hydrophobic properties, lipids cannot cross aqueous compartments without appropriate auxiliary components.
Phenomena of intracellular migration of molecules have
been investigated with various types of eukaryotic cells.
Several components involved in these processes were studied
at the molecular level, and some of the mechanisms are
understood already in some detail. Less attention has been
paid to the structural arrangement of organelles in connection to transport and communication processes. This aspect
appears to be specifically important for the assembly of
lipids into organelle membranes. Increasing recent evidence
suggested that membrane contact may be the most relevant
mechanism for lipid migration between organelles. This
mechanism was proposed to govern the import of lipids
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630
from the endoplasmic reticulum (ER) into mitochondria in
various experimental systems (for reviews see Daum &
Vance, 1997; Nebauer et al., 2003; Voelker, 2004, 2005;
Rosenberger & Daum, 2005), but also for the supply of
lipids from internal membranes to the plasma membrane
(Pichler et al., 2001; Baumann et al., 2005; Schnabl et al.,
2005; Sullivan et al., 2006).
To visualize membrane association and contact which
may be a prerequisite for translocation processes and/or
related cellular events we have used transmission electron
microscopy (TEM) for three-dimensional (3D) imaging of
the yeast Saccharomyces cerevisiae. TEM of ultrathin serial
sections of cells was originally used both for morphometric
measurements and evaluation of section profiles to obtain a
better view of organelles and cellular structures (Preuss
et al., 1992; Bähler et al., 1993; Mulholland et al., 1994; Sato
& Yano, 1994; Bennett & Laurie, 1995; Gaffal et al., 1995;
Manella et al., 1997; Singh et al., 1998; Yamaguchi et al.,
2002). Although various yeast species have already been
examined by ultrathin sectioning and computer assisted 3D
reconstructions, these studies were mostly restricted to the
visualization of the heterogeneity of mitochondrial structure
(Yamaguchi et al., 2003) or single selected cell structures
(Osumi, 1998; Winey et al., 2005). Recently, the 3D ultrastructure including basic quantitative data of the organelle
structure of the pathogenic yeast Exophiala dermatitidis was
investigated by ultrathin serial sectioning (Biswas et al.,
2003; Yamaguchi et al., 2003). 3D images of the internal
structural organization of Saccharomyces cerevisiae cells were
also obtained by X-ray tomography (Larabell & Le Gros,
2004) and using dual beam electron microscopy (Heymann
et al., 2006). The advantage of these methods is the
combination of fairly high resolution with a short time of
data collection. However, the resolution of these methods is
limited to c. 60 nm. Therefore internal membrane or organelle associations with distances of o 30 nm cannot be
identified. The reason for the paucity of such data may be
that 3D reconstruction of cells, organelles and fine structures
based on electron microscopic inspection of ultrathin serial
sections is still laborious and time consuming.
The method of 3D image reconstruction of yeast cells
employed in this study is similar to procedures reported
earlier (Stevens, 1977; Osumi, 1998; Zellnig et al., 2004). The
advantage of our method is that distinct structures of
interest can be shown separately while other elements like
organelles or their substructures are faded out. Selected
structures can be visualized from various angles, yielding
detailed information about association and arrangement of
organelles. For comparative reasons and to minimize misinterpretation due to fixation artifacts, both chemical fixation and cryofixation of yeast cells (Biswas et al., 2003;
Konomi et al., 2003; Feron et al., 2005; Winey et al., 2005;
Binns et al., 2006) were used in this study to obtain detailed
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A. Perktold et al.
data about organelle organization and membrane association in yeast.
Materials and methods
Strain and culture conditions
The haploid wild-type strain S. cerevisiae W303 was used
throughout this study. Cells were grown on YPD medium
containing 1% yeast extract, 2% peptone and 2% glucose to
the late logarithmic phase, harvested by centrifugation and
subjected to fixation as described below.
Fixation of cells and sectioning for electron
microscopy
Electron microscopic investigations were performed with
chemically fixed and high pressure frozen/freeze-substituted
material. For chemical fixation, cells were treated with 4%
paraformaldehyde/5% glutaraldehyde in 0.1 M cacodylate
buffer (pH 7.0) and 1 mM CaCl2 for 90 min at room
temperature. Then, cells were washed in buffer with 1 mM
CaCl2 for 1 h and incubated for 1 h with a 2% aqueous
solution of KMnO4. After washing for 30 min, samples were
dehydrated in a graded series of ethanol (50–100%, with en
bloc staining in 2% uranylacetate in 70% ethanol overnight)
and gradually infiltrated with increasing concentrations of
Spurr resin (30%, 50%, 70% and 100%) mixed with ethanol
for a minimum of 3 h of each step. Samples were finally
embedded in pure, fresh Spurr resin and polymerized at
60 1C for 48 h.
To prepare high pressure frozen (HPF) and freeze-substituted cells, pellets of yeast were high pressure frozen in the
Leica EM Pact (Leica Microsystems, Vienna, Austria). Platelets containing frozen samples were then transferred and
stored under liquid nitrogen conditions in transfer boxes or
prechilled specimen baskets. For freeze substitution (FS),
specimens were transferred into precooled cryogenic vials
(Corning Incorporated) filled with FS-medium consisting of
2% osmium tetroxide in anhydrous acetone containing
0.2% uranyl acetate. FS was carried out at 80 1C (72 h),
65 1C (24 h),
30 1C (24 h), 0 1C (12 h) and room
temperature (1 h). After rinsing the samples twice in anhydrous acetone for 15 min, they were infiltrated by solutions
containing acetone and Agar 100 epoxy resin (mixtures 2 : 1,
1 : 1, 1 : 2) and pure epoxy resin for at least 3 h at room
temperature. Embedded samples were then polymerized in
pure, fresh Agar 100 epoxy resin for 48 h at 60 1C.
Series of 80-nm ultrathin sections were cut with an
ultramicrotome (Reichert Ultracut S) and transferred to
single slot grids because their transmission area is high
enough to show a complete ribbon of serial ultrathin
sections without any disturbing bars. The ultrathin sections
FEMS Yeast Res 7 (2007) 629–638
631
Ultrastructural 3D imaging of the yeast
were stained with lead citrate and viewed with a Philips CM
10 transmission electron microscope (TEM). Section thickness restricts resolution in z direction to 80 nm.
Computer aided 3D reconstruction and
determination of surface areas and volumes
TEM micrographs of the serial sectioned yeast cells were
digitized by a scanner (Epson 4990 Photo) connected with a
Pentium IV computer (2.8 GHz, 1 GB RAM) and imported
as TIFF files. The digitized micrographs were pixel images
which were transferred into vectorgraphics by tracing
selected cell structures (organelles) semi-automatically
using a computer program (Corel Trace) or by hand (Corel
Draw). For 3D reconstructions, vectorgraphics were aligned
by centering specific structures. This step required visibility
of two section edges on selected micrographs, which allowed
general centering of images. If necessary, centering was
corrected by a one-upon-another arrangement of selected
ultrastructures of successive sections. 3D reconstructions
were created by the program CARRARA STUDIO (Softline).
Complete cells and organelles were measured and threedimensionally reconstructed. It is noteworthy that this
method led to images of reconstructed, real existing cell
structures and not to models based on statistical possibilities.
The circumference and area of sectioned organelles was
measured by the computer program OPTIMAS 6.5 (Bio Scan)
and data were exported to the software program EXCEL. The
total surface area and volume of organelles were calculated
by the sum of their circumferences/areas multiplied by the
section thickness. Organelle associations were determined
on the TEM micrographs by counting the number of
associations when two selected cell compartments (e.g.
mitochondria and ER) were at a distance of o 30 nm from
each other. The counting was performed on every single
section of the section series and resulted in the total number
of associations of two compartments within the whole cell.
As the number of associations may depend on the abundance and size of the organelles, data were also presented as
number of associations per 10 mm2 organelle surface area.
Results
General structural aspects
Conventional ultrathin sections of yeast cells were characterized by sectioned profiles of the major organelles such as
nucleus, mitochondria, vacuoles, endoplasmic reticulum
(ER) and lipid particles (Fig. 1a and c). It is, however,
impossible to judge the frequency of organelles and their
spatial relation from 2D images of the cell, or to assign
separate profiles of the same structure to a single or more
organelles of the same kind, e.g. vacuolar profiles in Fig. 1a.
FEMS Yeast Res 7 (2007) 629–638
In contrast, 3D reconstruction of cells provides information
about the number, arrangement and association of different
organelles and subcellular structures. Reconstructed images
of yeast demonstrated the dense organization of different
organelles (Fig. 1b and d). The large number of different
structures and organelles in the cell, however, made it almost
impossible to obtain a distinct view of individual structural
elements. An optical cut of the cell (Fig. 1b and d) or fading
out selected structures (Fig. 2) facilitated identification of
structural details. When cell wall and plasma membrane
were electronically subtracted from the image, a dense
network of peripheral ER became visible, especially in
chemically fixed cells (Fig. 2a). This subcellular structure is
often referred to as cortical ER, which is structurally
different from the perinuclear/nuclear ER (Prinz et al.,
2000; Voeltz et al., 2002; Estrada et al., 2005). The occurrence of ER fractions in proximity to the yeast plasma
membrane has been described before by Preuss et al. (1992)
and Prinz et al. (2000). It has to be noted, however, that the
peripheral ER did not completely shield internal organelles
from the plasma membrane because it was not a strict
continuum (Fig. 2a). In cryofixed cells (Fig. 2b) association
of the ER with the plasma membrane was less pronounced
than in chemically fixed cells. It appeared that chemical
fixation of yeast cells caused changes in the cellular distribution of the ER resulting in a delocalization towards the cell
periphery. Nevertheless, associations of the ER with the
plasma membrane could also be observed in cryofixed cells.
Besides the peripheral network, the ER was also found in
the interior of the cell where it came in contact with other
organelles. The latter observation became most evident
when other cell organelles were faded out (Figs. 2b–d). A
prominent type of organelle association occurred between
the ER and mitochondria. Physical interaction of these two
organelles from the yeast has been described before (Gaigg
et al., 1995; Achleitner et al., 1999). Mitochondria formed a
compact cell compartment consisting of 16–35 single organelles, which were at least in some cases found in close
vicinity to the ER (Fig. 2c and d).
The ER was also associated with lipid particles (Fig. 2b).
The physiological relevance of this observation has not yet
been proven. Studies from our laboratory (Leber et al., 1998;
Athenstaedt et al., 1999) and from others (Lum & Wright,
1995) had led to the assumption that lipid particles of the
yeast may be derived from the ER (for a recent review see
Athenstaedt & Daum, 2006). This hypothesis is in line with
the model of oil body biogenesis in plant cells (Napier et al.,
1996; Galili et al., 1998; Murphy & Vance, 1999; Zweytick
et al., 2000; Hills & Roscoe, 2006). Contact between yeast
lipid particles and the ER as shown in this study confirmed a
possible relationship between these two compartments.
Lipid particles of the yeast were often also found to be
attached to the vacuole (Fig. 2a and b). This observation is
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632
A. Perktold et al.
Fig. 1. Transmission electron micrographs of
ultrathin sections (a, c; bar = 1 mm) and corresponding 3D reconstructions of serial sections
(b, d) of a chemically fixed (a, b) and cryofixed
(c, d) yeast cell. (a, c) 2D section profiles of some
cell compartments. (b, d) the complexity of the
cell organelles is demonstrated by means of a
3D view. CW, cell wall; ER, endoplasmic reticulum; LP, lipid particle; M, mitochondrion; N,
nucleus; V, vacuole.
in line with cell fractionation studies (Zinser et al., 1991;
Leber et al., 1994), which showed that during sucrose
density gradient centrifugation crude vacuoles float to the
top of the gradient due to their attachment to lipid particles
(Uchida et al., 1988). Only upon treatment with EDTA is
this association destroyed; vacuoles can then be sedimented
like ‘regular’ organelles, whereas purified lipid particles still
float as a result of their low density.
The largest organelles of the yeast were vacuole and
nucleus, which were always found in close vicinity (Fig. 2e
and f). 3D reconstructions revealed that in chemically fixed
cells deformation of the two organelles had occurred (Fig.
2e). In cryofixed cells, the compact structured core of the cell
was protected and smoother (Fig. 2f) than in chemically
fixed cells. This picture most likely showed the true structural organization of nucleus and vacuole in the yeast cell due
to the cryo-preservation of the yeast cell in milliseconds.
Changes of the nuclear and vacuolar shape in chemically
fixed cells could mainly be attributed to the strongly reduced
volume of both organelles under these conditions, whereas
the surface area was only affected for vacuoles, as described
below in more detail.
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Association of organelles
3D reconstructions provided valuable information regarding the detailed structure of organelles from various angles.
These images allowed us to detect associations between
organelles, but also to quantify association rates. Such
calculations may be highly relevant for the evaluation of
transport or exchange rates of certain substances between
different compartments. We considered a distance of
o 30 nm between two organelles as an association event
due to the expected size of ‘contact proteins’ attached to or
associated with the membrane surface in the range of
4–45 nm (Achleitner et al., 1999). Such proteins may facilitate interaction between organelles as suggested by Corazzi
and coworkers (Rakowska et al., 1994; Camici & Corazzi,
1997; Corazzi et al., 1998), who described a microsomal
protein with potential fusogenic properties.
Associations between organelles of the yeast are numerous and may therefore be relevant for interorganelle communication which bypasses the cytoplasmic compartment
(Binns et al., 2006). Some organelles exhibit a specifically
high tendency to associate with each other. Based on 3D
FEMS Yeast Res 7 (2007) 629–638
633
Ultrastructural 3D imaging of the yeast
Fig. 2. 3D reconstructions of internal structures
of a chemically fixed (a, c, e) and cryofixed (b, d,
f) yeast cell showing selected organelles after
fading out of distinct cell structures. Square
= 1 mm2. (a, b) The cell wall and the plasma
membrane are faded out and the peripheral ER
system (green) becomes clearly visible in the
chemically fixed cell. (c, d) Arrangement and
association of the internal ER (green) and mitochondria (blue) after fading out of all other cell
structures. (e, f) Arrangement of the nucleus
(red) and vacuoles (purple) after fading out of all
other cell structures and rotation of the image in
(f). The fragmentation of the vacuole in the
chemically fixed cell (e) is clearly visible. ER,
endoplasmic reticulum; M, mitochondrion; N,
nucleus; V, vacuole.
imaging of yeast cells described in this study the frequency of
association between subcellular compartments was documented by counting membrane contacts per series of cell
sections (Table 1). Due to different surface areas of organelles, however, the number of associations per defined
organelle surface area was regarded as a more relevant
measure for the affinity between two compartments (Table
2). Both types of calculation were performed for chemically
fixed and cryofixed yeast cells. It has to be mentioned that all
data presented in this work were obtained from cells grown
on glucose. Other carbon sources may influence the cell
FEMS Yeast Res 7 (2007) 629–638
ultrastructure, the abundance of organelles and, consequently, association rates between subcellular compartments. Such observations, which suggested changes of the
cellular organization of yeast cells cultivated on fatty acids as
a carbon source resulting in the induction of peroxisomes,
were discussed in a previous paper from our laboratories
(Achleitner et al., 1999). More recent investigations (our
own unpublished results) confirmed this view.
Numerical data from cells treated with the two different
methods of fixation were in some cases dramatically different. The most prominent example was the calculated
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634
A. Perktold et al.
Table 1. Number of total associations of organelles (distance between
two organelles less than 30 nm) in yeast cells grown on YPD counted on
all serial sections of the cell
No. of associations in chemically/cryofixed cells
ER
M
N
V
LP
PM
ER
M
N
V
LP
PM
18/0
79/11
1/0
2/4
14/1
1139/10
79/11
4/7
0/4
0/2
0/4
0/1
1/0
0/4
0/0
22/3
35/2
0/0
2/4
0/2
22/3
15/0
32/1
0/0
14/1
0/4
35/2
32/1
2/3
0/0
1139/10
0/1
0/0
0/0
0/0
0/0
Data presented for chemically fixed/cryofixed cells.
ER, endoplasmic reticulum; LP, lipid particle; M, mitochondrion;
N, nucleus; PM, plasma membrane; V, vacuole.
Table 2. Total organelle surface area (mm2) and organelle volume (mm3)
per cell and corresponding number of associations (distance between
two organelles less than 30 nm) per 10 mm2 organelle surface area
ER
M
N
V
LP
PM
4/9
0.4/0.4
43/102
0/0
No. of associations per 10 mm2 organelle surface area
ER
1.9/0
2.9/1.2
0.1/0
0.1/0.4
3.8/0.4
M
2.9/1.2
1.4/2.2
0/0.6
0/0.2
0/1.4
N
0.1/0
0/0.6
0/0
6.1/0.4
46.1/1.1
V
0.1/0.4
0/0.2
6.1/0.4
7.9/0
42.1/0.3
LP
3.8/0.4
0/1.4
46.1/1.1 42.1/0.3
5.0/3.3
PM
28.5/0.4 0/0.03
0/0
0/0
0/0
28.5/0.4
0/0.03
0/0
0/0
0/0
0/0
Organelle data for chemically/cryofixed cells
93/28
29/32
19/20
19/35
mm2
mm3
0.7/0.4
2.4/1.7
2.6/5.3
1.9/14.1
Example of a calculation: 28.5 associations were calculated per 10 mm2
PM with 10 mm2 ER in a chemically fixed cell. Data presented for
chemically/cryofixed cells.
ER, endoplasmic reticulum; LP, lipid particle; M, mitochondrion;
N, nucleus; PM, plasma membrane; V, vacuole.
volume of nuclei and vacuoles from the two types of fixed
cells. Whereas cryofixation led to a calculated volume of
both organelles which roughly corresponds to a globular
shape of the organelle, chemical fixation dramatically decreased the volume values. Obviously, the latter treatment
led to massive shrinking of these large organelles. As can be
seen from Tables 1 and 2, however, the method of fixation
not only affected the shape of cell organelles as a result of
alterations in surface area and volume, but also the number
of organelle associations. Generally speaking, associations
between organelles were found to be more numerous in
chemically fixed cells than in cryofixed cells. In chemically
fixed cells the highest number of associations related to the
surface area was found between the organelle pairs lipid
particles/nucleus, lipid particles/vacuole, ER/plasma membrane and vacuole/nucleus. This calculation roughly
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reflected the relative number of associations between organelles (see Table 1) with the exception of ER/plasma
membrane and ER/mitochondria, whose single associations
appeared to be more frequent. With cryofixed cells the
relative number of ER/mitochondria associations (Table 1)
was similar to that of ER/plasma membrane. Other associations which were considered numerous in chemically fixed
cells were less pronounced in cryofixed cells. The calculated
ratios of associations per organelle surface area (Table 2)
demonstrated high association rates between lipid particles/
mitochondria, ER/mitochondria and lipid particles/nucleus.
Discussion
Contact between organelles has been recognized during the
last decade as important prerequisite for a number of
cellular processes. This surface contact of subcellular compartments does obviously not occur randomly, but appears
to be a directed event. Thus, it is not surprising that different
organelles associate with different efficiency as described in
this study. The molecular basis of organelle association may
be catalysis of fusogenic proteins, as has already been
suggested (Rakowska et al., 1994; Camici & Corazzi, 1997;
Corazzi et al., 1998). On the other hand, the occurrence of
‘contact blockers’ which may prevent association events
between subcellular compartments has to be taken into
account. It will be a task for the future to identify and
characterize such components, which may be highly important for communication and biochemical interaction
between organelles.
To visualize association between yeast organelles we have
chosen the method of 3D reconstruction of electron microscopic images derived from serial ultrathin sections, which
allowed structural investigations at the required level of
resolution. Whereas the method of computational reconstruction was well applicable for this purpose, the technique
of cell fixation turned out to be most critical. The choice of
the fixation method either by chemical treatment or by
cryotechnique strongly affected the shape of certain organelles as well as the occurrence of associations between
subcellular compartments. Although the general message of
these two experimental approaches appears to be the same,
namely that certain organelles such as mitochondria and the
ER, or the ER and the plasma membrane show a clear
preference for association, quantitative differences must not
be ignored. It appears that in chemically fixed cells swelling
or shrinkage of some organelles occurs, resulting in a much
higher number of associations than in cryofixed cells. The
organelles most strongly affected by the method of fixation
are the ER, the nucleus and the vacuole as shown by
calculation of their contacts to other organelles, their surface
area and their volume. Thus, although both fixation methods may be valuable for specific purposes, quantitative
FEMS Yeast Res 7 (2007) 629–638
635
Ultrastructural 3D imaging of the yeast
results should be interpreted with caution, and possible
effects of the fixation procedure leading to osmotic imbalance inside the cell have to be taken into account. Altogether,
cryofixation seems to yield a better and conserved view of
the native structure of the yeast than chemical fixation.
Visualization of organelle association does not, of course,
explain its biological role or relevance. It has been postulated
that contact between subcellular compartments may be
highly important for various cell dynamic processes. One
of the best studied organelle interactions is that of the ER
and mitochondria; this has also been the most prominent
association found in the present study. The possible role of
this contact in lipid migration between the two organelles
was reported before (Gaigg et al., 1995; Daum & Vance,
1997; Achleitner et al., 1999; Voeltz et al., 2002; Levine,
2004). A subfraction of the ER associated with mitochondria
from mammalian (Vance, 1990, 1991; Rusinol et al., 1994)
and yeast cells (Gaigg et al., 1995; Achleitner et al., 1999)
named MAM (mitochondria associated membrane) was
isolated and characterized. The MAM fraction of the yeast
was shown to be distinct from the bulk ER, especially
because of its high capacity to synthesize phospholipids.
Experiments with reconstituted isolated organelles (Gaigg
et al., 1995; Achleitner et al., 1999) or permeabilized cells
(Achleitner et al., 1995) demonstrated that contact between
MAM and mitochondria was a prerequisite for lipid transport between the two compartments. Schumacher et al.
(2002) showed that a yeast strain mutated in MET30, which
encodes a substrate recognition subunit of a multiprotein
SCF (Skip1/Cullin/F box protein components) ubiquitin
ligase, was affected in phosphatidylserine transport between
the two organelles. It has also been shown that inactivation
of Met4p by Met30p-mediated ubiquitination (Kaiser et al.,
1998), alleviated the inhibition of phosphatidylserine transport, possibly by disturbing membrane contact.
Another function that was ascribed to ER/mitochondria
contact, although not with the yeast, was Ca21 signaling and
regulation (Rizzuto et al., 1998; Csordas et al., 1999; Rutter
& Rizzuto, 2000; Filippin et al., 2003; Levine, 2004; Rutter,
2006). This process may be facilitated by enrichment of
IP3-activated receptors at the ER/mitochondria interfaces
(Rizzuto et al., 2004). Ca21-ions released from mammalian
ER in response to hormonal stimulation might be preferentially transferred into the mitochondrial matrix, causing
the local activation of ATP synthesis. The increase in
mitochondrial Ca21 might trigger a release of apoptosisactivating substrates and modify the activity of ER-located
Ca21 release channels and thus the dynamics of cytosolic
Ca21 oscillations.
Contact of the ER with organelles different from mitochondria, as also documented in this study, may be of equal
importance for short range nonvesicular intracellular trafficking (Levine, 2004). A subfraction of the yeast ER
FEMS Yeast Res 7 (2007) 629–638
associated with the plasma membrane might fulfill such
functions (Pichler et al., 2001). Similar to MAM, this
portion of the ER exhibited a high capacity to synthesize
lipids. An involvement in direct lipid transport to the
plasma membrane similar to the MAM/mitochondria system was postulated but not yet proven at the experimental
level. Transport of ergosterol from the ER to the plasma
membrane in yeast by a nonvesicular mechanism as shown
recently by Baumann et al. (2005) may occur through such
membrane contact zones. Intimate plasma membrane/ER
interactions have also been discussed as potentially essential
factors in the capacitative Ca21 entry in nonexcitable
mammalian cells (Putney, 1999; Yao et al., 1999; Patterson
et al., 1999). It was suggested that refilling of Ca21 stores of
the ER upon Ca21 release to the cytoplasm requires direct
interaction of the plasma membrane with the ER (Putney
et al., 2001).
In summary, the reason for the juxtaposition of the ER to
other organelles in the cell appears to be that all organelles
need components that are made or stored in the ER. For
mitochondria, which have no connection with the ER via
vesicular trafficking, direct transfer appears to be especially
important. In the case of the plasma membrane, traffic via
membrane contact may provide a selective alternative to
vesicle flux.
Another prominent organelle association is the nucleus–
vacuole junction (NV junction) which has been studied in
S. cerevisiae in some detail (for reviews see Voeltz et al., 2002;
Kvam & Goldfarb, 2006). NV junctions are created by
specific binding interactions between the vacuole membrane
protein Vac8p and the nuclear membrane protein Nvj1p
(Pan et al., 2000) forming a stabile complex. NV junctions
mediate piecemeal microautophagy of the nucleus (PMN),
an autophagic process that targets portions of the nucleus
for degradation in the vacuole (Roberts et al., 2003). At NV
junctions two proteins involved in lipid metabolism are
accumulated, namely Tsc13p and Osh1p (Levine & Munro,
2001; Kohlwein et al., 2001; Kvam & Goldfarb, 2004). Both
proteins are supposed to alter the local lipid composition of
the nuclear and vacuole membranes, thereby facilitating the
biogenesis of PMN vesicles (Kvam & Goldfarb, 2006).
Osh1p belongs to the yeast Osh family, which shows some
homology to mammalian oxysterol-binding protein (OSBP)
(Levine & Munro, 2001). OSBP and OSBP-related proteins
are cytoplasmic lipid-binding proteins and might specifically mediate lipid exchange at membrane contact sites
(Olkkonen & Levine, 2004).
In conclusion, examples of membrane contact discussed
in this study demonstrate the possible physiological relevance of organelle associations. Visualization and quantification of these associations on an ultrastructural level are
therefore important for the understanding and evaluation of
various cellular processes.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
636
Acknowledgements
This study was financially supported by the Fonds zur
Förderung der wissenschaftlichen Forschung in Österreich
(project 17321 to GD, and project P15374-B03 to GZ).
References
Achleitner G, Zweytick D, Trotter P, Voelker D & Daum G (1995)
Synthesis and intracellular transport of
aminoglycerophospholipids in permeabilized cells of the yeast,
Saccharomyces cerevisiae. J Biol Chem 270: 29836–29842.
Achleitner G, Gaigg B, Krasser A, Kainersdorfer E, Kohlwein SD,
Perktold A, Zellnig G & Daum G (1999) Association between
the endoplasmic reticulum and mitochondria of yeast
facilitates interorganelle transport of phospholipids through
membrane contact. Eur J Biochem 254: 545–553.
Athenstaedt K & Daum G (2006) The life cycle of neutral lipids:
synthesis, storage and degradation. Cell Mol Life Sci 63:
1355–1369.
Athenstaedt K, Zweytick D, Jandrositz A, Kohlwein SD & Daum
G (1999) Identification and characterization of major lipid
particle proteins of the yeast Saccharomyces cerevisiae.
J Bacteriol 181: 6441–6448.
Bähler J, Wyler T, Loidl J. & Kohli JJ (1993) Unusual nuclear
structures in meiotic prophase of fission yeast: a cytological
analysis. J Cell Biol 121: 241–256.
Baumann NA, Sullivan DP, Ohvo-Rekilä H, Simonot C, Pottekat
A, Klaassen Z, Beh CT & Menon AK (2005) Transport of newly
synthesized sterol to the sterol-enriched plasma membrane
occurs via nonvesicular equilibration. Biochemistry 44:
5816–5826.
Bennett MD & Laurie DA (1995) Chromosome size in maize and
sorghum using EM serial section reconstructed nuclei.
Maydica 40: 199–204.
Binns D, Januszewski T, Chen Y, Hill J, Markin VS, Zhao Y, Gilpin
C, Chapman KD, Anderson RGW & Goodman JM (2006) An
intimate collaboration between peroxisomes and lipid bodies.
J Cell Biol 173: 719–731.
Biswas SK, Yamaguchi M, Naoe N, Takashima T & Takeo K
(2003) Quantitative three-dimensional structural analysis of
Exophiala dermatitidis yeast cells by freeze-substitution and
serial ultrathin sectioning. J Electron Microsc 52: 133–143.
Camici O & Corazzi L (1997) Phosphatidylserine translocation
into brain mitochondria: Involvement of a fusogenic protein
associated with mitochondrial membranes. Mol Cell Biochem
175: 71–80.
Corazzi L, Monni M, Placidi M & Roberti R (1998) A
glycoprotein from rat liver endoplasmic reticulum promotes
both aggregation and fusion of liposomes and acidic pH.
J Membrane Biol 165: 53–63.
Csordas G, Thomas AP & Hajnoczky G (1999) Quasi-synaptic
signal transmission between endoplasmic reticulum and
mitochondria. EMBO J 18: 96–108.
Daum G & Vance J (1997) Import of lipids into mitochondria.
Prog Lipid Res 3: 103–130.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
A. Perktold et al.
Estrada P, Du Y, Novick P & Ferro-Novick S (2005) Ice2p is
important for the distribution and structure of the cortical ER
network in Saccharomyces cerevisiae. J Cell Sci 118: 65–77.
Feron G, Blin-Perrin C, Krasniewski I, Mauvais G & Lherminier J
(2005) Metabolism of fatty acid in yeast: characterization of
b-oxidation and ultrastructural changes in the genus
Sporidiobolus sp. cultivated on ricinoleic acid methyl ester.
FEMS Microbiology Letters 250: 63–69.
Filippin L, Magalhães PJ, Benedetto GD, Colella M & Pozzan T
(2003) Stable interactions between mitochondria and
endoplasmic reticulum allow rapid accumulation of calcium
in a subpopulation of mitochondria. J Biol Chem 278:
39224–39234.
Gaffal KP, Arnold CG, Friedrichs GJ & Gemple W (1995)
Morphodynamical changes of the chloroplast of
Chlamydomonas reinhardtii during the 1st round of division.
Arch Protistenkd 145: 10–23.
Gaigg B, Simbeni R, Hrastnik C, Paltauf F & Daum G (1995)
Characterization of a microsomal subfraction associated with
mitochondria of the yeast, Saccharomyces cerevisiae. Biochim
Biophys Acta 1234: 214–220.
Galili G, Sengupta-Gopalan C & Ceriotti A (1998) The
endoplasmic reticulum of plant cells and its role in protein
maturation and biogenesis of oil bodies. Plant Mol Biol 38:
1–29.
Heymann JAW, Hayles M, Gestmann I, Gianuzzi LA, Lich B &
Subramaniam S (2006) Site-specific 3D imaging of cells and
tissues with a dual beam microscope. J Struct Biol 155: 63–73.
Hills MJ & Roscoe TJ (2006) Synthesis of structural and storage
lipids by the ER. Plant Cell Monographs (4): The Plant
Endoplasmic Reticulum (Robinson DG, ed), pp. 155–186.
Springer-Verlag, Berlin-Heidelberg.
Kaiser P, Sia RA, Bardes EG, Lew DJ & Reed SI (1998) Cdc34 and
the F-box protein Met30 are required for degradation of the
Cdk-inhibitory kinase Swe1. Genes Dev 12: 2587–2597.
Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K, Bacikova D &
Daum G (2001) Tsc13p is required for fatty acid elongation
and localizes to a novel structure at the nuclear-vacuolar
interface in Saccharomyces cerevisiae. Mol Cell Biol 21:
109–125.
Konomi M, Fujimoto K, Toda T & Osumi M (2003)
Characterization and behaviour of a-glucan synthase in
Schizosaccharomyces pombe as revealed by electron microscopy.
Yeast 20: 427–438.
Kvam E & Goldfarb DS (2004) Nvj1p is the outer-nuclearmembrane receptor for oxysterol-binding protein homolog
Osh1p in Saccharomyces cerevisiae. J Cell Sci 117: 4959–4968.
Kvam E & Goldfarb DS (2006) Nucleus vacuole junctions in
yeast: anatomy of a membrane contact site. Biochem Soc Trans
34: 340–342.
Larabell CA & Le Gros MA (2004) X-ray tomography generates
3-D reconstructions of the yeast, Saccharomyces cerevisiae, at
60-nm resolution. Mol Biol Cell 15: 957–962.
FEMS Yeast Res 7 (2007) 629–638
637
Ultrastructural 3D imaging of the yeast
Leber R, Zinser E, Zellnig G, Paltauf F & Daum G (1994)
Characterization of lipid particles of the yeast, Saccharomyces
cerevisiae. Yeast 10: 1421–1428.
Leber R, Landl K, Zinser E, Ahorn H, Spök A, Kohlwein SD,
Turnowsky F. & Daum G (1998) Dual localization of squalene
epoxidase, Erg1p, in yeast reflects a relationship between
endoplasmic reticulum and lipid particles. Mol Biol Cell 9:
375–386.
Levine T (2004) Short-range intracellular trafficking of small
molecules across endoplasmic reticulum junctions. Trends Cell
Biol 14: 483–490.
Levine TP & Munro S (2001) Dual targeting of Osh1p, a yeast
homologue of oxysterol-binding protein, to both the Golgi
and the nucleus-vacuole junction. Mol Biol Cell 12: 1633–1644.
Lum PL & Wright R (1995) Degradation of HMG-CoA reductaseinduced membranes in the fission yeast, Schizosaccharomyces
pombe. J Cell Biol 131: 81–94.
Manella C, Marko M & Buttle K (1997) Reconsidering
mitochondrial structure: new views of an old organelle. Trend
Biochem Sci 22: 37–38.
Mulholland J, Preuss D, Moon A, Wong A, Drubin D & Botstein
D (1994) Ultrastructure of the yeast actin cytoskeleton and its
association with the plasma membrane. J Cell Biol 125:
381–391.
Murphy DJ & Vance J (1999) Mechanisms of lipid-body
formation. Trends Biochem Sci 24: 109–115.
Napier JA, Stobart AK & Shewry PR (1996) The structure and
biogenesis of plant oil bodies: the role of the ER membrane
and the oleosin class of proteins. Plant Mol Biol 31: 945–956.
Nebauer R, Birner R & Daum G (2003) Biogenesis and cellular
dynamics of glycerophospholipids in the yeast Saccharomyces
cerevisiae. Topics Curr Genet 6: 125–168.
Olkkonen VM & Levine TP (2004) Oxysterol binding protein: in
more than one place at one time? Biochem Cell Biol 82: 87–98.
Osumi M (1998) The ultrastructure of yeast: cell wall structure
and formation. Micron 29: 207–233.
Pan X, Roberts P, Chen Y, Kvam E, Shulga N, Huang K, Lemmon
S & Goldfarb DS (2000) Nucleus-vacuole junctions in
Saccharomyces cerevisiae are formed through the direct
interaction of Vac8p with Nvj1p. Mol Biol Cell 11: 2445–2457.
Patterson RL, van Rossum DB & Gill DL (1999) Store-operated
Ca21 entry: Evidence for a secretion-like coupling model. Cell
98: 487–499.
Pichler H, Gaigg B, Hrastnik C, Achleitner G, Kohlwein SD,
Zellnig G, Perktold A & Daum G (2001) A subfraction of the
yeast endoplasmic reticulum associates with the plasma
membrane and has a high capacity to synthesize lipids.
Eur J Biochem 268: 2351–2361.
Preuss D, Mulholland J, Franzusoff A, Segev N & Botstein D
(1992) Characterization of the Saccharomyces Golgi complex
through the cell cycle by immunoelectron microscopy.
Mol Biol Cell 3: 789–803.
Prinz WA, Grzyb L, Veenhuis M, Kahana JA, Silver PA. &
Rapoport TA (2000) Mutants affecting the structure of the
FEMS Yeast Res 7 (2007) 629–638
cortical endoplasmic reticulum in Saccharomyces cerevisiae.
J Cell Biol 150: 461–474.
Putney Jr JW (1999) Kissin’ cousins: intimate plasma membraneER interactions underlie capacitative calcium entry. Cell 99:
5–8.
Putney Jr JW, Broad LM, Braun FJ, Lievremont JP & Bird GS
(2001) Mechanisms of capacitative calcium entry. J Cell Sci
114: 2223–2229.
Rakowska M, Zborowski J & Corazzi L (1994) A fusogenic
protein from rat microsomal membranes: partial purification
and reconstitution into liposomes. Membr Biol 142: 35–42.
Rizzuto R, Duchen MR & Pozzan T (2004) Flirting in little space:
The ER/Mitochondria Ca21 liaison. Sci STKE 215, doi:
10.1126/stke.2152004re1.
Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz
LM, Tuft RA & Pozzan T (1998) Close contacts with the
endoplasmic reticulum as determinants of mitochondrial
Ca21 responses. Science 280: 1763–1766.
Roberts P, Moshitch-Moshkovitz S, Kvam E, O’Toole E, Winey M
& Goldfarb DS (2003) Piecemeal microautophagy of the
nucleus in Saccharomyces cerevisiae. Mol Biol Cell 14: 129–141.
Rosenberger S & Daum G (2005) Lipids of mitochondria.
Research Signpost 1735: 111–118.
Rusinol AE, Cui Z, Chen MH & Vance JE (1994) A unique
mitochondria-associated membrane fraction from rat liver has
a high capacity for lipid synthesis and contains pre-Golgi
secretory proteins including nascent lipoproteins. J Biol Chem
269: 27494–27502.
Rutter GA (2006) Moving Ca21 from the endoplasmic reticulum
to mitochondria: is spatial intimacy enough? Biochem Soc
Trans 34: 351–355.
Rutter GA & Rizzuto R (2000) Regulation of mitochondrial
metabolism by ER Ca21 release: an intimate connection.
Trends Biochem Sci 25: 215–212.
Sato S & Yano H (1994) The nucleolonema changes its threedimensional conformation during interphase in root-tip
meristems of onion. Protoplasma 179: 172–180.
Schnabl M, Daum G & Pichler H (2005) Multiple lipid transport
pathways to the plasma membrane in yeast. Biochim Biophys
Acta 1687: 130–140.
Schumacher MM, Choi JY & Voelker DR (2002)
Phosphatidylserine transport to the mitochondria is regulated
by ubiquitination. J Biol Chem 277: 51033–51042.
Singh S, Lazzaro MD & Walles B (1998) The nuclear reticulum in
placental cells of Lilium regale is a part of the endomembrane
system. Protoplasma 203: 144–152.
Stevens BJ (1977) Variation in number and volume of the
mitochondria in yeast according to growth conditions. A study
based on serial sectioning and computer graphics
reconstitution. Biol Cellulaire 28: 37–56.
Sullivan DP, Ohvo-Rekila H, Baumann NA, Beh CT & Menon AK
(2006) Sterol trafficking between the endoplasmic reticulum
and plasma membrane in yeast. Biochem Soc Trans 34:
356–358.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
638
Uchida E, Ohsumi Y & Anraku Y (1988) Purification of yeast
vacuolar membrane H1-ATPase and enzymological
discrimination of three ATP-driven proton pumps in
Saccharomyces cerevisiae. Methods Enzymo 157: 544–562.
Vance JE (1990) Phospholipid synthesis in a membrane fraction
associated with mitochondria. J Biol Chem 265: 7248–7256.
Vance JE (1991) Newly made phosphatidylserine and
phosphatidylethanolamine are preferentially translocated
between rat liver mitochondria and endoplasmic reticulum.
J Biol Chem 266: 89–97.
Voelker DR (2004) Genetic analysis of intracellular
aminoglycerophospholipid traffic. Biochem Cell Biol 82: 156–169.
Voelker DR (2005) Bridging gaps in phospholipid transport.
Trends Biochem Sci 30: 396–404.
Voeltz GK, Rolls MM & Rapoport TA (2002) Structural
organization of the endoplasmic reticulum. EMBO Reports 3:
944–950.
Winey M, Morgan GP, Straight PD, Giddings Jr TH &
Mastronarde DN (2005) Three-dimensional ultrastructure of
Saccharomyces cerevisiae meiotic spindles. Mol Biol Cell 16:
1178–1188.
Yamaguchi M, Biswas SK, Suzuki Y, Furukawa H, Sameshima M
& Takeo K (2002) The spindle pole body duplicates in early G1
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
A. Perktold et al.
phase in the pathogenic yeast Exophiala dermatitidis: an
ultrastructural study. Exp Cell Res 279: 71–79.
Yamaguchi M, Biswas SK, Suzuki Y, Furukawa H & Takeo K
(2003) Three-dimensional reconstruction of a pathogenic
yeast Exophiala dermatitidis cell by freeze-substitution and
serial sectioning electron microscopy. FEMS Micobiol Lett 219:
17–21.
Yao Y, Ferrer-Montiel AV, Montal M & Tsien RY (1999)
Activation of store-operated Ca21 current in Xenopus oocytes
requires SNAP-25 but not a diffusible messenger. Cell 98:
475–485.
Zellnig G, Zechmann B & Perktold A (2004) Morphological
and quantitative data of plastids and mitochondria within
drought stressed spinach leaves. Protoplasma 223:
221–227.
Zinser E, Sperka-Gottlieb CDM, Fasch EV, Kohlwein SD, Paltauf
F & Daum G (1991) Phospholipid synthesis and lipid
composition of subcellular membranes in the unicellular
eukaryote, Saccharomyces cerevisiae. J Bacteriol 173:
2026–2034.
Zweytick D, Athenstaedt K & Daum G (2000) Intracellular lipid
particles of eukaryotic cells. Biochim Biophys Acta 1469:
101–120.
FEMS Yeast Res 7 (2007) 629–638