Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844; and * Department of Biochemistry, University of Washington, Seattle, Washington 98195
Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as IB2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.
Cell proliferation depends on the inheritance of organelles, which are duplicated and segregated into
daughter cells rather than synthesized de novo
during cell division (reviewed in Warren and Wickner, 1996 Like vacuole inheritance (Haas and Wickner, 1996 Vacuole inheritance also requires LMA1, a novel heterodimeric protein which contains thioredoxin (Xu and
Wickner, 1996 We now report that the other subunit of LMA1 is IB2, a
previously described S. cerevisiae protein that can inhibit
vacuolar proteinase B (Maier et al., 1979 Reagents and Strains
Reagents were purchased or prepared as described (Xu and Wickner,
1996 In Vitro Homotypic Vacuole Fusion Assay
Preparation of cytosol, vacuoles, salt-washed vacuoles, and purification of
the p10 subunit of LMA1 were as described (Xu and Wickner, 1996 The 30-µl in vitro vacuole fusion reaction (Haas et al., 1994 Gel Filtration
Cytosol (stored at Microscopy
Yeast vacuoles were visualized with the vital fluorophores fluorescein-5isothiocyanate (FITC, Molecular Probes, Inc., Eugene, OR) or FM 4-64 as
described (Vida and Emr, 1995 Other Methods
Protein concentration was determined with a protein reagent (Bio-Rad
Laboratories, MA) with BSA as a standard. SDS-PAGE and "High Tris"
SDS-PAGE (Xu and Wickner, 1996 The vacuole inheritance reaction can be assayed in vitro
by incubating isolated, salt-washed vacuoles with ATP and
cytosol at physiological temperatures. The reaction is performed with vacuoles purified from two yeast strains (Haas
et al., 1994 The Small Subunit of LMA1
We have previously reported the purification of a low molecular weight activity, termed LMA1, which supports the in vitro reaction (Xu and Wickner, 1996
Table I.
IB2 Is Needed for Efficient Vacuole Inheritance In Vivo
).
Through cytological, genetic, and biochemical means, we
have begun to study the inheritance of vacuoles (lysosomes) in Saccharomyces cerevisiae. In early S phase, the
vacuole projects a stream of vesicles and membranous tubules, termed a segregation structure, into the bud (Weisman and Wickner, 1988
; Gomes de Mesquita et al., 1991;
Raymond et al., 1992
). These vesicles fuse in the bud,
forming the daughter cell vacuole. Vacuole inheritance is defective in vac mutants in which the growing bud receives
little or no maternal vacuolar material (Weisman et al.,
1990
; Shaw and Wickner, 1991
; Nicolson et al., 1995
). This
process can be studied in an in vitro vacuole inheritance
assay. In the presence of ATP, cytosol, and physiological
temperature, isolated vacuoles form segregation structures
and undergo homotypic fusion (Conradt et al., 1992
, 1994;
Haas et al., 1994
). The fusion can be monitored by fluorescence microscopy and by biochemical assays. This reaction is abolished when its components are prepared from vac
mutants, establishing its authenticity (Haas et al., 1994
;
Nicolson et al., 1995
). This in vitro vacuole inheritance reaction requires Sec18p and Sec17p (Haas and Wickner,
1996
), Ypt7p (Haas et al., 1995
), and a low molecular
weight activity termed LMA1 (Xu and Wickner, 1996
).
), many
vesicle trafficking and membrane fusion events employ
N-ethylmaleimide-sensitive fusion protein (NSF)1, and soluble NSF attachment proteins (SNAPs) (Rothman, 1994
). The homologues of NSF and
-SNAP in budding yeast are
Sec18p and Sec17p, respectively (Wilson et al., 1989
; Griff
et al., 1992
). NSF is a homotrimeric ATPase that requires
SNAPs to bind to membranes at SNAP receptors, termed
SNAREs (Söllner et al., 1993
). NSF supports membrane
trafficking events such as vesicular transport from the ER
through the Golgi apparatus to the endosomes and plasma membrane, homotypic endosome fusion, and synaptic vesicle fusion to the plasma membrane (Rothman, 1994
). Although homotypic ER fusion and nuclear envelope fusion
require neither NSF nor SNAP, they require Cdc48, an
NSF-like protein (Latterich et al., 1995
). In yeast, Sec17p
and Sec18p work together to promote the ATP-dependent release of Sec17p from the vacuole membrane at an early
stage of the in vitro inheritance reaction (Mayer et al., 1996
).
). Thioredoxin is a ubiquitous small protein
(Holmgren, 1985
) with a pair of cysteine within a highly
conserved active site. These cysteine residues are reduced
by an NADPH-dependent thioredoxin reductase and, in
turn, reduce target proteins. Although thioredoxin function generally involves its redox properties, redox-independent roles are also known (Russel and Model, 1986
;
Huber et al., 1986
; Tonissen et al., 1993). Deletion of both
yeast thioredoxin genes obliterates LMA1 activity of the
cytosol in vitro and causes a striking vacuole inheritance
defect in vivo (Xu and Wickner, 1996
).
; Schu et al., 1991
).
Both components of LMA1 are required for efficient vacuole inheritance. LMA1 acts via a novel mechanism as, in
this reaction, thioredoxin is not employing a redox mechanism and IB2 is not acting via inhibition of proteinase B. LMA1 and Sec18p synergistically support fusion of saltwashed vacuoles and act in an ordered manner at an early
stage of the in vitro vacuole inheritance reaction.
Materials and Methods
; Mayer et al., 1996
; Haas and Wickner, 1996
). Yeast strains were described in Xu and Wickner (1996)
and Muller (1995)
. The strains YS18
(Mata ura3-5 his3-11 leu2-3,-112 canR) and YPS19 (Mata
pbi2::URA3)
were kindly provided by Dr. D.H. Wolf (Institut für Biochemie der Universitat Stuttgart, Germany).
). For
salt-washing, freshly isolated vacuoles were diluted to 0.3 mg/ml with 0%
Ficoll buffer (Haas et al., 1994
). Equal volumes of BJ3505 and DKY6281
vacuoles were mixed and KCl and KOAc added (from 3M stocks) to 100 mM and 50 mM, respectively. Aliquots of 200 µl were transferred to Eppendorf tubes, incubated 10 min at 30°C, chilled at 0°C for 2 min, and centrifuged (10,000 g, 75 s, 4°C). Supernatants were removed and pellets covered with 160 µl of cold 0% Ficoll buffer. LMA1 and Sec18p were largely removed by salt-washing.
) contains
4.8 µg of mixed, salt-washed vacuoles from BJ3505 and DKY6281, 20 mM
Pipes-KOH, pH 6.8, 200 mM sorbitol, 150 mM KOAc, 5 mM MgCl2, an
ATP regenerating system (1 mM ATP, 40 mM creatine phosphate, and 70 U/ml creatine phosphokinase), either 1 mg/ml cytosol (from yeast strain
K91-1A), fractions from Sephacryl S100HR gel filtration or purified proteins as indicated, and 0.1× proteinase inhibitor cocktail (PIC; Xu and
Wickner, 1996
). Salt-washed vacuoles were used in all experiments. In vitro reactions were incubated at 25°C for 90-120 min and stopped by chilling on ice. Alkaline phosphatase activity was determined as described
(Haas et al., 1994
). 1 U of fusion activity corresponds to 1 µmol of p-nitrophenol produced at 25°C per minute per microgram of BJ 3505 vacuoles.
80°C) was thawed and mixed with PIC and ATP to final concentrations of 30× and 0.1 mM, respectively, and then incubated
on ice for 20 min followed by centrifugation (14,000 g, 15 min, 4°C). A
200-µl aliquot of clarified cytosol was applied to a Sephacryl S100HR column (1 cm × 26 cm) equilibrated in buffer C (20 mM TrisCl, pH 8.3, 5 mM
Mg(OAc)2, and 0.1 mM MnCl2). This buffer has been optimized in Mn
concentration for LMA1 purification. The column was eluted (12 ml/cm2/h,
300 µl fractions) with buffer C at 4°C.
; Xu and Wickner, 1996
).
) and affinity purification of IgG and
immunoblot analysis (Mayer et al., 1996
; Haas and Wickner, 1996
) have
been described. Thioredoxin reductase was purified from baker's yeast as
described (Gonzales et al., 1970). Reductase activity was determined with
the 5,5
-dithiobis (2-nitrobenzoic acid) (DTNB) assay described in Luthman and Holmgren (1982).
Results
). One strain has normal vacuolar proteases
but a deletion in the PHO8 gene encoding the vacuole proalkaline phosphatase. The other strain has a normal PHO8
gene but lacks vacuolar proteases A and B which are
needed for processing the catalytically inactive pro-alkaline phosphatase to its mature, active form. Upon fusion of
vacuoles isolated from these strains, the proteases from
one fusion partner cleave the pro-alkaline phosphatase
from the other to produce the catalytically active enzyme,
allowing quantitative assay of vacuole fusion.
). LMA1 is a 23-kD heterodimer with a thioredoxin subunit of 12 kD and a smaller
subunit termed p10. Edman degradation of the purified p10
subunit showed that it has the sequence ThrLysAsnPheIleValThrLeuLysLysAsnThrProAspValGluAlaLysLys which
is identical to a portion of the reported sequence of yeast proteinase yscB inhibitor IB2, encoded by the PBI2 gene
(Schu et al., 1991
). Antibody prepared to a peptide from the
COOH terminus of this protein specifically decorated the
p10 subunit of LMA1 (data not shown), confirming its identification from the NH2-terminal sequence. To determine whether IB2 is a functional component of LMA1, cytosols
were prepared from a pbi2 null mutant and its corresponding wild-type strain. Cytosolic components were resolved by Sephacryl S100HR chromatography and fractions were assayed for their ability to replace cytosol in the
in vitro reaction of vacuole fusion. The wild-type cytosol showed both the previously reported (Xu and Wickner,
1996
) high molecular weight activity peak, termed HMA,
and LMA1 (Fig. 1 A). Deletion of the PBI2 gene left the
HMA peak while obliterating activity from the LMA1 region (Fig. 1 B). The identity of the LMA1 peak from wildtype cytosol was confirmed by immunoblot with anti-TRX1p
peptide antiserum, whereas monomeric thioredoxin, recovered from more included fractions of pbi2
cytosol (data not
shown), had no activity in our assay (Fig. 1 B). Deletion of
the PBI2 gene had no effect on the cellular content of
thioredoxin (data not shown). Thus IB2 is clearly an essential component of LMA1. To test whether IB2 plays a role
in vacuole inheritance in vivo, the PBI2 deletion mutant
and wild-type cells were examined by fluorescence microscopy. The PBI2 deletion mutant showed a clear vac phenotype of buds which frequently had no vacuole (Fig. 2 B).
While almost all wild-type cells (Fig. 2 A) with a bud diameter of at least half the maternal cell diameter had inherited a bud vacuole, 34% of PBI2 deletion cells had no vacuole in their buds (Fig. 2 B and Table I). Taken together,
these in vitro and in vivo results demonstrate that IB2, previously identified as a cytosolic inhibitor of vacuolar proteinase B, is a novel factor required for efficient vacuole inheritance in yeast.
Fig. 1.
Deletion of PBI2 abolishes LMA1 activity. Yeast cytosols were prepared from PBI2 (A) and pbi2 (B) strains (Schu
et al., 1991
) and fractionated by Sephacryl S100HR gel filtration
as described in Materials and Methods. Aliquots (4 µl) were assayed for protein concentration (open circles) and fusion activity
(closed circles) as described in Materials and Methods. LMA2 is
for unknown reasons not present in the PBI2 wild-type strain
background.
[View Larger Version of this Image (53K GIF file)]
Fig. 2.
Vacuole inheritance deficiency in pbi2 cells. Wildtype (A) or
pbi2 (B) yeast were grown in YCM medium (Rogers and Bussey, 1978) to early log phase. Cells from 1 ml of the
culture were harvested (3,000 g, 5 min, 4°C) and resuspended in
1 ml YCM with 10 µl of 4 mg/ml FITC (Molecular Probes Inc.,
Eugene, OR). Cells were incubated at 37°C for 10 min, collected
by centrifugation, resuspended in 50 µl of YCM, and examined
by fluorescence microscopy. Bar, 4 µm.
[View Larger Version of this Image (59K GIF file)]
Mechanism of LMA1 Action
To understand how LMA1 participates in the in vitro reaction, we tested whether it requires the redox activity of its
thioredoxin subunit or the proteinase B inhibitor activity
of its IB2 subunit. Yeast thioredoxin reductase was assayed
and purified from baker's yeast as described (Gonzales et al.,
1970; Luthman and Holmgren, 1982). No stimulation of
fusion was observed when both NADPH and purified
thioredoxin reductase were added to fusion reactions supported by LMA1 (Fig. 3 A). To further examine the role of
the thioredoxin redox activity in vacuole fusion, we used a
mutant form of Trx1p in which both cysteinyl residues at
its active site were replaced by serines (Muller, 1995). The
redox activity of Trx1p is completely abolished by this
means. The genes for mutant and wild-type forms of
TRX1 were transformed into the trx1, trx2 double deletion
strain and cytosols were prepared. As reported (Xu and
Wickner, 1996
), the fusion activity of TRX1,TRX2 wildtype cytosol (Fig. 3 B a) is resolved into HMA, LMA1,
and LMA2 by Sephacryl S100HR gel filtration and the
LMA1 peak is selectively lost in the trx1,trx2 double deletion strain (Fig. 3 B b). As expected, the LMA1 peak is restored when the wild-type TRX1p is expressed from a
plasmid in the trx1,trx2 double deletion strain (Fig. 3 B c).
Strikingly, the LMA1 peak is also restored in this trx1,trx2
double deletion strain by a plasmid which expresses the
double Cys to Ser mutant form of Trx1p (Fig. 3 B d). Furthermore, the vac phenotype of the trx1,trx2 double deletion strain was rescued in vivo by introducing this double
Cys to Ser mutant form of Trx1p (data not shown). These
data demonstrate that the function of thioredoxin in vacuole inheritance and fusion does not employ a cyclic reduction-oxidation mechanism.
Since LMA1 does not act via redox, we asked whether it
acts by inhibiting vacuolar proteinase B, the purported activity of its other subunit. Sec18p is one of the two necessary soluble factors, as shown below (Fig. 5), and functions
with LMA1 to support vacuole fusion and is also protease
sensitive. We therefore checked the stability of Sec18p
during the fusion reaction in the presence, or absence, of
LMA1. The amount of soluble and membrane-bound fulllength Sec18p remained unchanged (Fig. 4 A), indicating
that LMA1 is not acting to prevent Sec18p degradation.
To determine whether LMA1 functions in vacuole fusion
via inhibition of proteinase B cleavage of some other, unidentified vacuole protein, we prepared vacuoles from the
strain BJ3505 (Moehle et al., 1986) in which both proteinase A and B genes are deleted and examined the fusion of
these vacuoles by fluorescence microscopy. While clustering of these vacuoles was promoted by Sec18p (Mayer and
Wickner, 1997), the fusion of these protease-deficient vacuoles still requires both LMA1 and Sec18p (Fig. 4 B).
Measurements of all vacuole diameters in random fields
showed average diameters of 0.58 µm (0.25 SEM), 0.73 µm
(0.24), 0.66 µm (0.22), or 1.29 µm (0.21) after incubation with ATP only, LMA1 only, Sec18p only, or LMA1 and
Sec18p, respectively. Thus, LMA1 does not support vacuole fusion via a proteinase B inhibitor activity of its IB2
subunit. The function of LMA1 in vacuole fusion must employ novel mechanisms which are unrelated to the established functions of its subunits in other cellular processes.
Purified LMA1 and Sec18p Synergistically Support In Vitro Fusion of Salt-washed Vacuoles
Both LMA1 and Sec18p can support vacuole fusion (Xu
and Wickner, 1996; Haas and Wickner, 1996
), yet the degree of requirement for each of these pure proteins varies
among fresh vacuole preparations (Xu, Z., A. Mayer, E. Muller, and W. Wickner, unpublished observations). We
find a consistent requirement for both proteins when the
vacuoles have been salt-washed. Thus, little fusion occurred with limiting concentrations of Sec18p or LMA1
alone (Fig. 5 A), but full activity was obtained when both
proteins were added. Furthermore, the reaction supported
by these two pure proteins occurred to the same extent as
seen with cytosol (Fig. 5 B) and was still sensitive to
Gdi1p, GTP
S, and microcystin-LR, known inhibitors of
later stages of the reaction (Conradt et al., 1994
; Haas et
al., 1994
, 1995). Our isolated vacuole preparations have
variable amounts of Sec18p and LMA1 that are largely removed or inactivated during a salt-wash procedure, and
these two pure proteins constitute a minimal set of soluble
proteins needed for reconstitution of the fusion reaction.
LMA1 Action Follows that of Sec18p and Sec17p Proteins
Previous studies (Conradt et al., 1994) have established
that our in vitro reaction of vacuole inheritance occurs in
defined stages in an obligatory sequence. To establish the
stage(s) where Sec18p and LMA1 act, vacuoles were incubated with ATP, LMA1, and Sec18p. At the indicated
times (Fig. 6 A), vacuoles were collected by centrifugation
and resuspended in either the same, complete reaction mix
or in buffer and ATP alone before resuming the second incubation. To control for fusion that occurred at each time
of centrifugation, an aliquot was also transferred to ice.
After a 30-min incubation, the reisolated vacuoles can undergo fusion without added LMA1 or Sec18p in the second incubation (Fig. 6 A). As expected, the reaction became resistant to anti-Sec18p antibodies in the second
incubation but was still sensitive to GTP
S and Microcystin LR (Fig. 6 B). Thus, LMA1 and Sec18p participate at
an early stage of the reaction.
To determine whether Sec18p and LMA1 function in an
obligatory sequence, we first tested whether LMA1 stimulated the Sec17p release reaction, driven by Sec18p and
ATP. Vacuoles were incubated with either no addition,
LMA1, Sec18p, or both proteins for various times, then reisolated and assayed by immunoblot for bound Sec17p. LMA1 does not stimulate Sec17p release in our assay (Fig.
7 A). We further examined Sec17p release from fresh vacuoles (not salt-washed) prepared from a trx1, trx2 double
deletion strain and a pbi2 null strain and found that neither LMA1 subunit is required to stimulate Sec17p release
(data not shown). We then determined whether LMA1 action can be completed before that of Sec18p. Vacuoles
were incubated with ATP and LMA1 alone for 30 min, after which time LMA1 can normally be removed without
loss of subsequent reaction (Fig. 6). After reisolation, vacuoles still required both LMA1 and Sec18p for fusion (Fig.
7 B). Thus, the requirement for LMA1 cannot be satisfied
before the Sec18p-catalyzed release of Sec17p.
LMA1 cannot act long after Sec18p-mediated Sec17p
release, as this reaction creates a labile state of the vacuoles in which the vacuoles remain intact (data not shown)
but lose competence for the reaction which leads to fusion
(Fig. 8 A). Vacuoles were pre-incubated with ATP alone
or with ATP and either Sec18p, antibody to Sec18p, or
LMA1. After various incubation times, vacuoles were collected and resuspended in reaction buffer with ATP and
both LMA1 and Sec18p for a second incubation. In agreement with earlier observations (Conradt et al., 1994), the
competence of vacuoles to undergo fusion was lost during
pre-incubation in the absence of LMA1 (Fig. 8 A) or cytosol
(data not shown); the latter may contain additional stabilizing factors as well as LMA1. This lability was exacerbated by added Sec18p and partially relieved by an antibody to Sec18p and thus was largely due to Sec18p action.
This lability limits the interval after Sec18p-mediated
Sec17p release in which LMA1 can act. Can the Sec18p/
Sec17p reaction be separated from the LMA1 requirement, despite their close kinetic relationship? We incubated vacuoles with Sec18p for varying intervals, mixed them with an antibody to Sec17p, and added LMA1 (Fig.
8 B). Upon addition at 0 time, the antibody effectively
blocked the reaction as there had been insufficient time
for Sec17p release (Mayer et al., 1996
). If the vacuoles
were instead incubated for a few minutes before addition
of antibody to Sec17p, the release reaction occurred with
little vacuole inactivation and LMA1, when added, "captured" the activated vacuoles for the subsequent fusion reaction. After longer incubations, however, the labile vacuoles
(Fig. 8 A) became inactive. This delay in addition of LMA1
resulted in loss of subsequent fusion capacity (Fig. 8 B).
Thus LMA1 must act during, or shortly after, the Sec18p/
17p reaction to permit the later steps of the reaction.
Our in vitro reaction measures the last step of inheritance,
the fusion of vacuoles, but relies on a sequence of coupled
steps which represents earlier stages in the pathway as
well. Thus, the first step of inheritance to be observed in
vivo, the creation of a segregation structure which projects
into the bud, is also seen in our in vitro reaction (Conradt et
al., 1992) and requires the vac-encoded proteins (Conradt et
al., 1992
; Haas et al., 1994
; Nicolson et al., 1995
). Recent
studies (Fanton, V., and W. Wickner, unpublished) indicate
that structure formation requires Sec17 and Sec18 proteins, further connecting it to the early stages of the in
vitro reaction as assayed biochemically (Mayer et al., 1996
). Analysis of the in vitro reaction thus offers insight
into several stages of the organelle inheritance process.
Analysis of this inheritance reaction initially established
four subreactions which occur in an obligate order: (I) A
brief salt-dependent step, (II) a cytosol-dependent step,
(III) an ATP-dependent step, and (IV) a step sensitive to
microcystin-LR (Conradt et al., 1994). These reactions culminate in the fusion of vacuoles and mixing of contents,
assayed by proenzyme maturation. Subsequent studies
have identified some of the proteins which support these reactions. These include Sec17p (
-SNAP) and Sec18p
(NSF) (Haas and Wickner, 1996
), LMA1 (Xu and Wickner,
1996
), and the Ras-like GTPase Ypt7p (Haas et al., 1995
).
As isolated, the vacuoles bear on their surface many or, in
some vacuole preparations, all of the requisite proteins.
Nevertheless, after "salt-wash," both Sec18p and LMA1
are needed to reconstitute the cytosol requirement. Like
the earlier findings with cytosol, our current studies show that these proteins act at an early stage. LMA1 stabilizes
the reaction competence of vacuoles and is required during or immediately after the Sec18p-mediated release of
Sec17p. Docking also requires prior Sec17p release (Mayer
et al., 1997) and is followed by the Stage IV (Conradt et al.,
1994
) microcystin LR and GTP
S-sensitive fusion reaction.
Despite the increasing resolution of this reaction into an
ordered series of steps, the catalytic roles of the relevant
proteins are not known. LMA1, the focus of the current
report, was detected in a general scheme of cytosol fractionation and purified to homogeneity (Xu and Wickner,
1996). Each subunit, thioredoxin and IB2, is needed for
normal vacuole inheritance in vivo (Fig. 2 and Table I;
also, Xu and Wickner, 1996
). However, LMA1 acts neither in a typical thioredoxin redox reaction (Fig. 3) nor
solely to inhibit proteinase B (Fig. 4). Thioredoxin has
previously been shown to be required in a nonredox fashion in T7 replication (Huber et al., 1986
) where it confers
processivity on the polymerase, in filamentous phage assembly (Russel and Model, 1986
), and in the mammalian
early pregnancy factor system (Tonissen et al., 1993). It is
also reasonable that the IB2 subunit has a role in addition
to that of proteinase B inhibitor, since IB2 is cytosolic and
proteinase B is soluble in the vacuole lumen and thus
these two proteins may only meet infrequently under normal growth conditions. Though our current studies indicate that LMA1 may stabilize the activity of vacuoles in
early reaction steps, the LMA1 requirement cannot be
satisfied before Sec18p action. LMA1 acts during or immediately after the Sec17p/Sec18p reaction; its relation to
vacuole docking and to the Ypt7p-dependent events is still
to be determined. Nevertheless, the observation of its coupling to the action of the yeast homologues of NSF and
-SNAP, which are required for almost every step of membrane trafficking, suggests that LMA1 or its functional
equivalents may also have a necessary role in other intracellular trafficking events. A detailed comparison of homotypic fusion during vacuole inheritance and the heterotypic fusion events during inter-organelle vesicular traffic
will require identification of any SNARE proteins which support vacuole fusion.
Further understanding of the mechanism of vacuole inheritance will require a full resolution of the proteins which
support the process. This in turn raises the question of developing assays for these components. Some assays will
arise from a continued definition of subreactions, such as
searching for the early salt-modulated factor, the components needed for Sec17p release, the agents of docking, or
the functional receptors for vacuole association of LMA1,
Sec17p, Sec18p, and Ypt7p. Antibodies from the burgeoning yeast community may provide a second source of assays, just as they have allowed us to establish the roles of
several components. A third source of assays is the growing collection of vac mutants (Wang et al., 1996) which may
allow in vitro complementation. Continued fractionation
of the cytosol will also yield components, from the HMA
and LMA2 activities (Slusarewicz, P., Z. Xu, and A. Haas,
unpublished) to kinases and phosphatases which may regulate inheritance components (Conradt et al., 1992
, 1994). Finally, with as many individual components as possible in
hand, the vacuole membrane will have to be solubilized
and reconstituted into proteoliposomes which are competent for the individual subreactions.
Received for publication 10 October 1996 and in revised form 21 November 1996.
Address all correspondence to W. Wickner, Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844. Tel.: (603) 650-1701. Fax: (603) 650-1353.We thank Dr. Dieter H. Wolf for generously providing yeast strains, Drs. Albert Haas, Pawel Slusarewicz, Robert Matts, and Charles Barlowe for discussions, and Marilyn Leonard and Barbara Atherton for expert assistance.
This work was supported by a grant from NIGMS.
NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein.