Department of Physiology, 615 Michael St, 605G Whitehead Building, Emory University School of Medicine, Atlanta, GA 30322, USA
Author for correspondence (e-mail: wnichols{at}physio.emory.edu )
Accepted 1 April 2002
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Summary |
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Key words: Phosphatidylcholine, Phosphatidylethanolamine, Vacuole, Fluorescence, Yeast
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Introduction |
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Much of the trafficking involved in the generation of the plasma membrane's
unique lipid composition is thought to take place in the Golgi apparatus
because its lipid composition reflects properties of both the ER and the
plasma membrane (Zambrano et al.,
1975). In addition to helping establish interorganellar
differences in lipid composition, the Golgi is a prime example of
intraorganellar lipid organization. If the membranous stacks of the cis- and
trans-Golgi are compared, there is a dramatic increase in the content of
cholesterol as well as sphingolipids in the trans-Golgi network, making these
membranes more plasma membrane-like (Orci
et al., 1981
).
Another example of intraorganellar lipid organization is at the plasma
membrane of eukaryotic cells. The evidence currently available is consistent
with aminophospholipids being sequestered to the cytoplasmic leaflet of the
membrane, whereas choline lipids are enriched in the outer leaflet (reviewed
by Devaux and Zachowski,
1994). It is well established that loss of this asymmetric
distribution, resulting in exposure of PtdSer in the outer leaflet of the
plasma membrane, is required for activation of the coagulation cascade and can
serve as a signal for clearance of aging red blood cells and apoptotic cells
(reviewed by Zwaal and Schroit,
1997
). Despite these advances in our understanding of the
existence and significance of non-random lipid distributions within cells, the
dynamic processes involved in generating and maintaining this level of
organization remain poorly understood.
Historically, fractionation studies have proven informative by establishing
the steady-state distribution of lipids both between and within organelles.
However, these approaches do not address the dynamics of lipid movement. To
investigate such issues, a variety of additional tools have been developed
including short-chain, labeled lipids (reviewed by
Daleke and Lyles, 2000). These
lipid analogs have proven useful for studying lipid transport and trafficking
events. For example, in many cell lines, phosphatidylethanolamine (PtdEtn) and
PtdSer analogs are internalized rapidly by flip whereas phosphatidylcholine
(PtdCho) is internalized by flip slowly, if at all (reviewed by
Devaux and Zachowski, 1994
;
Zwaal and Schroit, 1997
).
These dynamic differences are thought to account for the asymmetric
distribution of endogenous phospholipids at the plasma membrane. Furthermore,
synthetic lipids appear to be sorted in a backbone and head-group specific
manner within cells. In fact, fluorescent labeled ceramide is commonly used as
a Golgi marker (Lipsky and Pagano,
1985b
) and can be metabolized into other fluorescent sphingolipid
analogs (Lipsky and Pagano,
1985a
) indicating that this fluorescent probe is a suitable
substrate for endogenous lipid modifying enzymes.
To study the establishment and maintenance of inter- and intra-organellar
lipid organization, we have examined the trafficking of fluorescent labeled
phospholipids in the genetically tractable yeast, Saccharomyces
cerevisiae. In our initial studies we found that short-chain
fluorescent-labeled 1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
aminocaproyl]-phosphatidyl-ethanolamine (M-C6-NBD-PE) is taken up
by yeast at normal growth temperature and labels predominantly the
mitochondria and nuclear envelope/endoplasmic reticulum without being
metabolized to an appreciable extent (Kean
et al., 1997). By contrast,
1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) predominantly
labels the lumen of the vacuole where it is catabolized
(Kean et al., 1993
). In
pep4 mutants, which do not process vacuolar hydrolases to their
active form, M-C6-NBD-PC still labels the lumen of the vacuole, but
its catabolism is dramatically inhibited, indicating that intact
M-C6-NBD-PC is trafficked to the lumen of the vacuole prior to its
breakdown (Kean et al.,
1993
).
These and other observations led to the hypothesis that
M-C6-NBD-PE was internalized by a non-endocytic mechanism,
presumably protein-mediated, inward-directed flip across the plasma membrane,
whereas M-C6-NBD-PC was thought to be internalized by endocytosis
and subsequently trafficked to the vacuole
(Kean et al., 1993). However,
our more recent studies showed that mutants defective for the internalization
step of endocytosis are not defective for the net uptake of
M-C6-NBD-PC or M-C6-NBD-PE
(Grant et al., 2001
). These
data, combined with the fact that yeast can internalize both lipids to a
similar extent at 2°C, when vesicular trafficking is inhibited, support
the model that M-C6-NBD-PC as well as M-C6-NBD-PE are
internalized predominantly by a non-endocytic mechanism, presumably
inward-directed transbilayer translocation or flip, across the plasma membrane
(Grant et al., 2001
;
Hanson and Nichols, 2001
).
Furthermore, we have shown that internalization of both M-C6-NBD-PC
and M-C6-NBD-PE is dependent on the plasma membrane proton
electrochemical gradient and is inhibited in anterograde sec mutants,
suggesting that flip of these phospholipid analogs across the plasma membrane
requires continuous transport of a proton-coupled flippase to the cell surface
(Grant et al., 2001
).
In addition to decreasing fluorescent lipid internalization we found that
one sec mutant, sec18, inhibited vacuolar localization of
M-C6-NBD-PC (Grant et al.,
2001). Because SEC18 is the yeast homologue of the
mammalian NEM-sensitive factor (NSF) required for membrane fusion in numerous
vesicle trafficking steps (Burd et al.,
1997
; Graham and Emr,
1991
; Hicke et al.,
1997
), we hypothesized that vesicular traffic is necessary for
vacuolar localization of M-C6-NBD-PC. Having ruled out endocytosis
as the primary means for M-C6-NBD-PC uptake, we examined which of
the remaining vesicular trafficking steps were required for
M-C6-NBD-PC sorting to the vacuole and showed that trafficking from
the pre-vacuolar compartment to the vacuole is necessary for its access to the
lumen of the vacuole. Furthermore, we demonstrated that addition of even a
single methyl group to the M-C6-NBD-PE head group was sufficient
for its sorting to the vacuole.
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Materials and Methods |
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Yeast strains and cultures
The S. cerevisiae strains used in this study are shown in
Table 1. All
temperature-sensitive strains were assayed for the inability to grow at
37°C on YPAD (1% yeast extract, 2% peptone, 0.004% adenine, 2% glucose)
plates prior to lipid trafficking assays. For mutants without a
temperature-sensitive growth defect, other mutation-related phenotypes, such
as the mislocalization of FM4-64 were confirmed. For all experiments, early
log-phase cultures (OD600=0.2-0.4) were grown in SDC [0.67% yeast
nitrogen base, 2% glucose, and complete amino acid supplement, as described
(Sherman et al., 1986)] from
overnight cultures. SCNaN3 is SDC media lacking glucose but
containing 2% sorbitol and 20 mM sodium azide. SCNaN3 + NaF is SDC
media lacking glucose but containing 2% sorbitol, 10 mM sodium fluoride and 10
mM sodium azide. Temperature-sensitive strains were grown at a permissive
temperature of 23°C and internalization assays were performed at either
23°C or 37°C, as indicated.
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Lipid preparation
To prepare dimethyl sulfoxide-solubilized stocks, aliquots of
NBD-phospholipids dissolved in chloroform were dried under a stream of
nitrogen, desiccated under vacuum for at least 1 hour, and resuspended in the
appropriate amount of DMSO to obtain a 1000x stock concentration. To
prepare large unilamellar vesicles, phospholipids dissolved in chloroform were
mixed in a 4:6 mole ratio of NBD-phospholipid to dioleoylphosphatidylcholine,
dried under a stream of nitrogen, and desiccated under vacuum for at least 1
hour. Desiccated phospholipids were resuspended by vortex mixing with SDC and
the mixture was passed 5-8 times through a Lipex Extruder (Lipex Biomembranes,
Vancouver, BC, Canada) equipped with 0.1 µm filters to produce a solution
of vesicles with 1 mM total phospholipid. Phospholipid preparations were made
fresh for each experiment.
Synthesis of monomethyl and dimethyl M-C6-NBD-PE
Monomethyl M-C6-NBD-PE (1-myristoyl-2-[6-(NBD)
aminocaproyl]-phosphatidylmonomethylethanolamine) and dimethyl
M-C6-NBD-PE (1-myristoyl-2-[6-(NBD)
aminocaproyl]-phosphatidyldimethylethanolamine) were synthesized from
M-C6-NBD-PE by phospholipase D-catalyzed based exchange
(Comfurius and Zwaal, 1977).
M-C6-NBD-PE was reacted with phospholipase D from cabbage (Sigma)
in the presence of either 2-dimethylethanolamine or 2-(methylamino)ethanol
(Acros Organics). Reaction products were purified by preparative TLC.
Appropriate bands were identified by their predicted mobility in relation to
M-C6-NBD-PE and M-C6-NBD-phosphatidate standards,
scraped, and eluted into chloroform/methanol. Purified products yielded a
single spot by TLC.
Internalization of phospholipid and dyes into yeast cells
For analysis of wild-type and deletion strains at ambient temperature,
cultures were grown to early log phase in SDC at 30°C. DMSO-solubilized
lipid was added to a final concentration of 5 µM and vortex mixed. After
45-minute incubation with the lipid at 30°C, cells were harvested and
washed twice with SDC at room temperature. To label mitochondrial and nuclear
DNA, the yeast were resuspended in 1 ml of SDC, and 1 µl of DAPI was added
from a 1 mg/ml stock solution in water. After 15 minutes of incubation with
DAPI at 30°C, cells were washed three times with ice-cold
SCNaN3 and imaged by fluorescence microscopy.
For labeling experiments performed at 2°C, cells were grown to early log phase in SDC and labeled with DAPI as described above. Yeast were then cooled to 2°C for 15 minutes prior to the addition of lipid. Lipid was added to a final concentration of 20 µM by pipetting an aliquot from the stock solution while vortex mixing the cells. After addition of lipid, cells were incubated at 2°C for 90 minutes. Cells were then either washed with ice-cold SCNaN3 or treated as described in Fig. 4 legend.
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For labeling experiments performed on temperature-sensitive strains, the mutants and their isogenic parental strains were grown to early-log phase in SDC at 23°C. The incubation temperature was then shifted to 37°C for 30 minutes prior to the addition of phospholipid vesicles to a final concentration of 50 µM total phospholipid. Cells were incubated for 45 minutes with the vesicles before adding DAPI to a final concentration of 1 µg/ml. After an additional 15 minute incubation with both DAPI and NBD-phospholipids, the cells were washed three times with two volumes of ice-cold SCNaN3 and imaged by fluorescence microscopy.
Fluorescence microscopy
Fluorescence microscopy was performed on a Zeiss Axiovert 135 microscope
equipped with barrier filters that eliminate detectable crossover of NBD and
DAPI fluorescence. The fluorescent images were enhanced with a SIT66
image-intensifying camera (DAGE-MTI, Michigan City, IN), digitized, and stored
using Metamorph software (Universal Imaging).
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Results |
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Vacuolar localization of M-C6-NBD-PC is not mediated by
endocytosis from the plasma membrane
The localization of M-C6-NBD-PC to the vacuole is one of several
pieces of evidence that led to the hypothesis that M-C6-NBD-PC is
internalized by endocytosis. However, upon further examination, we found that
strains defective for the internalization step of endocytosis were not
defective in the uptake of M-C6-NBD-PC or M-C6-NBD-PE
(Grant et al., 2001). Here we
show that, in addition to being unaffected in the net uptake of
NBD-phospholipids, end4 mutants
(Raths et al., 1993
) display
no defect in sorting M-C6-NBD-PC to the vacuole
(Fig. 2). These data indicate
that the standard pathway for endocytosis and recycling of plasma membrane
components is not required for the uptake or proper localization of
M-C6-NBD-PC.
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NBD-PC is not trafficked to the vacuole at low temperature
Although significantly slower, the non-endocytic mechanism of
M-C6-NBD-PC and M-C6-NBD-PE uptake presumably
inward-directed transbilayer transfer or flip still functions when
cells are incubated at low temperature, even on ice
(Grant et al., 2001;
Hanson and Nichols, 2001
).
However, under these conditions M-C6-NBD-PC is not trafficked to
the vacuole (Grant et al.,
2001
). In fact, at low temperature, M-C6-NBD-PC and
M-C6-NBD-PE exhibit the same labeling pattern, with both lipids
enriched in the mitochondria, nuclear envelope/endoplasmic reticulum and
perhaps other intracellular organelles
[Fig. 3
(Grant et al., 2001
;
Hanson and Nichols, 2001
)].
This labeling pattern is thought to be achieved through a passive mechanism
because it is not disrupted in respiration-defective petite strains that
exhibit dramatic reductions in mitochondrial membrane potential
(Hanson and Nichols,
2001
).
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Because these fluorescent phospholipids have a shortened acyl chain at the sn-2 position, it is possible that once flipped to the inner leaflet of the plasma membrane, M-C6-NBD-phospholipids can passively diffuse through the cytosol and eventually be enriched in the cytoplasmic leaflet of membranes with the most favorable partitioning properties. This default labeling pattern can, of course, be disrupted if the cell actively sorts some lipids into the lumen of an organelle, thereby effectively trapping even short-chain lipids.
M-C6-NBD-PC is trafficked from intracellular membranes to
the vacuolar lumen by a vesicle-mediated, energy-dependent mechanism
Given that our data indicate that the early stages of endocytosis are not
required for the internalization and distribution of M-C6-NBD-PC to
the vacuole (Fig. 2), we
predicted that cells in which the intracellular organelles were labeled with
M-C6-NBD-PC at 2°C would traffic this probe to the vacuole upon
warming to 30°C. The images presented in
Fig. 4 demonstrate that this is
in fact the case. Following labeling of the nuclear envelope/ER, mitochondria,
and perhaps other undetermined intracellular membranes with
M-C6-NBD-PC at 2°C, cells were warmed to 30°C for 60
minutes and imaged. During the 30°C chase period M-C6-NBD-PC is
trafficked to the vacuole lumen (Fig.
4A, left panel). As this trafficking occurs, the cell-associated
fluorescence becomes noticeably dimmer for two reasons some of the
lipid is effluxed out of the cell and either degraded or washed away
(Kean et al., 1997), and the
lipid that is trafficked to the vacuole is degraded to water-soluble products
with low quantum yield (Monti et al.,
1977
; Nichols,
1987
). Traffic to the vacuole, as well as efflux across the plasma
membrane (Hanson and Nichols,
2001
), is blocked upon warming, when the cells are depleted of
cellular energy by incubation with azide- and fluoride-containing media
following the internalization period and during the 30°C chase period
(Fig. 4A, right panel).
Given that M-C6-NBD-PC internalized at low temperature, in
addition to its traffic to the vacuole, is effluxed across the plasma membrane
and degraded, it is possible that the NBD fluorescence observed in the vacuole
results from the endocytosis of M-C6-NBD-PC or its degradative
products from the plasma membrane. To address this possibility, the warm-up
experiment was repeated in an end4 strain, which is blocked in
both fluid-phase and receptor-mediated endocytosis from the plasma membrane
(Wesp et al., 1997
). A
commonly used marker of endocytosis, FM4-64
(Vida and Emr, 1995
), was used
to confirm defective endocytosis in the mutant strain. The images in
Fig. 4B illustrate that
M-C6-NBD-PC is trafficked to the vacuole upon warming following low
temperature labeling in both the end4
and its isogenic parent
strain. However, FM4-64 is internalized to the vacuole only in the parent and
not the end4
strain. Thus, following its internalization to
intracellular membranes at low temperature, M-C6-NBD-PC is
trafficked independently from M-C6-NBD-PE to the vacuole by an
energy-dependent mechanism that does not require endocytosis from the plasma
membrane.
Previous results demonstrated that a mutant strain carrying a
temperature-sensitive, loss-of-function mutation in the SEC18 gene
does not traffic M-C6-NBD-PC to the vacuole
(Grant et al., 2001).
SEC18 encodes the yeast homologue of the NEM-sensitive fusion
protein, NSF, which is required for fusion of vesicles with target membranes
at many sites throughout the cell (Burd et
al., 1997
; Graham and Emr,
1991
; Hicke et al.,
1997
). These data suggest that at least one of the
energy-dependent steps in M-C6-NBD-PC traffic to the vacuole is
dependent on NSF-mediated vesicle fusion.
Class E vps mutants do not sort M-C6-NBD-PC to the
vacuole
Having established that sorting of M-C6-NBD-PC to the vacuole
was mediated by vesicular trafficking, but not endocytosis from the plasma
membrane, we next hypothesized that M-C6-NBD-PC was traveling
through the vacuolar protein sorting (vps) pathway in order to reach the lumen
of the vacuole and that its topological reorientation from the cytoplasmic to
lumenal compartments was a necessary step at some point along the pathway. A
logical place for this to occur would be at the ER. Because
M-C6-NBD-PC readily labels the nuclear envelope/ER, an organelle
that is known to exhibit flippase activity in yeast
(Nicolson and Mayinger, 2000)
as well as in mammalian cells (Bishop and
Bell, 1985
), this lipid could be preferentially flipped from the
cytoplasmic leaflet to the lumen of the ER and subsequently shunted through
the vps pathway to the vacuole. To test whether this occurred exclusively in
the ER, we examined the labeling pattern of a sec12-4 mutant that,
when incubated at the non-permissive temperature, is defective in the guanine
nucleotide exchange activity required for vesicular traffic from the ER to the
Golgi apparatus (Nishikawa and Nakano,
1993
; Novick et al.,
1980
). If flip across the ER membrane was the only site for
M-C6-NBD-PC to enter the lumenal compartment of the vps pathway,
then blocking ER to Golgi vesicle traffic would prevent its vacuolar
localization. As seen in Fig.
5A, vacuolar localization of M-C6-NBD-PC is not
perturbed in the sec12-4 mutant at the restrictive temperature. Thus,
although M-C6-NBD-PC flip is likely to occur across the ER membrane
(Nicolson and Mayinger, 2000
),
we concluded that neither flip across the ER membrane nor ER to Golgi traffic
is required for M-C6-NBD-PC transport to the vacuole.
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The next major vesicular trafficking step in the vps pathway is from the
Golgi to the pre-vacuolar compartment (PVC), and this event can be disturbed
by disruption of the VPS8 gene
(Horazdovsky et al., 1996). If
M-C6-NBD-PC entered the vps pathway exclusively by flip into the
lumen of the Golgi and subsequent traffic to the vacuole, then disruption of
Golgi to PVC traffic would prevent vacuolar labeling by
M-C6-NBD-PC. This hypothesis seemed plausible based on the reported
flippase activity in the Golgi apparatus
(Chen et al., 1999
). However,
as seen in Fig. 5B, the lumen
of the vacuole is brightly labeled in strains disrupted at the VPS8
locus, which indicates that neither Golgi to PVC vesicle traffic nor flip into
the Golgi lumen is necessary for vacuolar localization of
M-C6-NBD-PC.
The final vesicular trafficking step in the vps pathway is from the PVC to
the vacuole itself. Class E vps mutants are defective in this membrane
trafficking event and exhibit an exaggerated PVC full of small vesicles
(Raymond et al., 1992;
Rieder et al., 1996
;
Wurmser and Emr, 1998
). These
vesicles are thought to result from invaginations of the PVC membrane that are
pinched off and subsequently trafficked to the vacuole, where they are
degraded (Wurmser and Emr,
1998
). In fact, this mode of lipid turnover is hypothesized to be
the major mechanism for PtdIns(3)P degradation
(Wurmser and Emr, 1998
). Thus,
the invagination of vesicles and/or flippase activity at the PVC provide two
potential mechanisms for the topological reorientation necessary for
M-C6-NBD-PC traffic to the vacuolar lumen. If either or both of
these mechanisms were present in the PVC, but not in the vacuole, class E
mutants would not exhibit M-C6-NBD-PC vacuolar labeling. In fact,
this was observed. Fig. 5C
illustrates that M-C6-NBD-PC is not localized in the vacuole, but
instead is enriched in the mitochondria of a strain carrying a disruption in
the vps4 gene which encodes an AAA ATPase essential for PVC to
vacuole trafficking (Finken-Eigen et al.,
1997
). In addition, another class E mutant disrupted at the
VPS28 locus (Rieder et al.,
1996
) exhibited mitochondrial rather than vacuolar labeling by
M-C6-NBD-PC (data not shown).
Monomethyl and dimethyl M-C6-NBD-PE are sorted to the
vacuole
Given the intriguing differences between the trafficking of
M-C6-NBD-PC and M-C6-NBD-PE, lipids that differ
structurally by only three methyl groups, we chose to examine the trafficking
of monomethyl and dimethyl M-C6-NBD-PE the NBD analogues of
the two intermediates in the methylation pathway for PtdCho synthesis from
PtdEtn (Kanipes and Henry,
1997). As seen in Fig.
6, both monomethyl and dimethyl M-C6-NBD-PE label the
vacuole suggesting that even a single methylation event is sufficient for
targeting of PtdEtn to the vacuole. Since M-C6-NBD-PE is not
converted to detectable amounts of NBD-labeled monomethyl PtdEtn, dimethyl
PtdEtn, or PtdCho in wild-type cells (Kean
et al., 1997
), it is unlikely that the monomethyl and dimethyl
analogues are first converted to M-C6-NBD-PC prior to traffic to
the vacuole. However, to be certain that this was not occurring, we repeated
these experiments in a mutant strain in which both PtdEtn methyl transferase
genes, PEM1/CHO2 (Greenberg et
al., 1983
; Kodaki and
Yamashita, 1987
) and PEM2/OPI3
(Kodaki and Yamashita, 1987
;
Summers et al., 1988
), were
deleted and found the same result (data not shown). Thus, it is unlikely that
traffic of the NBD-labeled monomethyl and dimethyl PtdEtn to the vacuole
depends on their metabolism to M-C6-NBD-PC, and we concluded that
the sorting mechanism that distinguishes PtdEtn from PtdCho for transport can
also recognize the monomethyl and dimethyl ethanolamine head groups.
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Discussion |
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Although methylation has long been recognized as a modifier of protein
function (reviewed by Aletta et al.,
1998), few reports, if any, have indicated that methylation can so
dramatically alter the trafficking of membrane lipids. However, as with any
study employing labeled lipids, extrapolation to the movement of endogenous
lipids must be made with caution. Clearly, endogenous PtdCho will be subject
to a different set of trafficking `rules' because its two long acyl chains
will prevent it from exchanging freely between membranous organelles in the
absence of vesicular traffic or phospholipid transfer proteins. Regardless,
the ability of cells to target lipids with methylated amine head-groups for
degradation in the vacuole could provide a highly specific and efficient means
for maintaining proper PtdCho or lyso-PtdCho homeostasis within the cell, as
well as a pathway for recycling choline under conditions of choline
deprivation (Walkey et al.,
1998
). The selective targeting of the methylated products of
PtdEtn to the vacuole for degradation appears to be analogous to the finding
that functional PVC to vacuole trafficking is required for the proper turnover
of PtdIns(3)P (Wurmser and Emr,
1998
).
Analysis of vacuolar localization of M-C6-NBD-PC in mutants
defective in various vesicular trafficking events throughout the vps pathway
demonstrated that trafficking from the PVC to the vacuole is the only step
required for proper localization. The observed block of M-C6-NBD-PC
traffic to the vacuole in the class E vps mutants
(Fig. 5C) identifies the vps
pathway as the predominant mode of M-C6-NBD-PC traffic to the
vacuole, since protein traffic via the alternative ALP pathway bypasses the
PVC and is not inhibited in class E mutants
(Cowles et al., 1997;
Piper et al., 1997
). However,
the observed block of M-C6-NBD-PC vacuolar transport in the class E
vps mutants does not rule out the possibility that M-C6-NBD-PC can
move from a cytoplasmic topological location to the lumen of organelles at
prior steps in the vps pathway. Although the exact mechanism of
M-C6-NBD-PC movement to the lumen of the vacuole has yet to be
determined, the requirement for trafficking from the PVC to the vacuole would
appear to rule out the direct flip or invagination of M-C6-NBD-PC
into the vacuole. However, a block of vesicular traffic from the PVC to the
vacuole might preclude the transport of a vacuolar flippase or proteins
required for invagination to their proper location in the vacuole, thereby
preventing M-C6-NBD-PC uptake from the cytosolic compartment into
the lumen of the vacuole. If this were true, mutants defective in trafficking
from the ER to Golgi (sec12-4) or from the Golgi to PVC
(vps8
) should also block traffic of the newly synthesized
essential proteins to the vacuole and thus also block M-C6-NBD-PC
vacuolar labeling. Since M-C6-NBD-PC is trafficked to the vacuole
in both the sec12 and vps8
mutant strains
(Fig. 5A,B), we excluded the
possibility that M-C6-NBD-PC is internalized directly into the
vacuole.
The diagram presented in Fig.
7 summarizes the steps involved in trafficking
M-C6-NBD-PC from the plasma membrane to the vacuole and illustrates
two alternative models for the internalization of M-C6-NBD-PC into
the lumen of the PVC. In the first model, M-C6-NBD-PC and
methylated M-C6-NBD-PE gain access to the lumenal topological
compartment of the vps pathway via a methylated PtdEtn-specific flippase in
the PVC. In the second model, invagination of the PVC as described by Emr and
colleagues (Rieder et al.,
1996; Wurmser and Emr,
1998
) is proposed to explain the movement of
M-C6-NBD-PC from a cytoplasmic topology into the lumen of the
vacuole. According to this model, the PVC continually forms invaginations that
are subsequently pinched off as intra-PVC vesicles
(Rieder et al., 1996
;
Wurmser and Emr, 1998
). These
small vesicles are then packaged into larger vesicles that are trafficked to
the vacuole for degradation (Wurmser and
Emr, 1998
). Under this paradigm, M-C6-NBD-PC could
insert into the outer leaflet of the PVC and get trapped within a vesicle
formed by invagination. As part of this vesicle, M-C6-NBD-PC would
then be trafficked to the lumen of the vacuole and degraded
(Fig. 7).
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Both models are consistent with the observations that
M-C6-NBD-PC trafficking to the vacuole is blocked in the class E
vps mutants, vps4 and vps28
, which are blocked
in vesicular transport from the PVC to the vacuole, and the sec18-1
mutant, which is blocked at multiple vesicular trafficking steps. If the PVC
invagination model were accurate, one might expect to see
M-C6-NBD-PC labeling of an exaggerated PVC in the class E vps
mutants. Although this labeling pattern was observed occasionally (data not
shown), most cells exhibited M-C6-NBD-PC enrichment in the nuclear
envelope/ER and mitochondria, as seen in
Fig. 5B. The lack of consistent
labeling of an exaggerated PVC in the class E mutants remains unclear,
although it may reflect a requirement for continued vesicular flux through the
PVC for the internalization of M-C6-NBD-PC.
The data presented here do not distinguish the PVC-localized flippase model
from the invagination model. However, the data clearly demonstrate the
existence of a sorting process that distinguishes PtdCho and its methylated
precursors from PtdEtn. This head group-dependent sorting may either be
achieved by a specific binding site on the flippase or by a molecular sieve
inherent in the budding machinery that allows selective lateral diffusion of
methylated PtdEtn products into the emerging bud. Furthermore, both mechanisms
may be functionally interdependent in that an energy-dependent invagination
process might require selective flippase activity to accommodate the geometric
constraints inherent in producing small diameter buds, or conversely, an
energy-dependent flippase activity might generate the unequal surface pressure
between the two leaflets of the PVC bilayer required to drive the invagination
of small buds (Devaux and Zachowski,
1994). In addition to clarifying this mechanistic ambiguity, it
remains to be determined whether the primary function of selective vacuolar
transport and degradation of PtdCho is metabolic, in that it results in the
selective turnover and recycling of choline perhaps for reincorporation into
PtdCho via the CDP-choline pathway, or whether its transport and degradation
are byproducts of its primary function as an essential step in the membrane
invagination and budding process.
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Acknowledgments |
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References |
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