Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.

Abstract

Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.

Full text

Page 1

T H E J O U R N A L O F C E L L B I O L O G Y
©
The Journal of Cell Biology, Vol. 171, No. 5, December 5, 2005 823–833
http://www.jcb.org/cgi/doi/10.1083/jcb.200505145
The Rockefeller University Press$8.00
JCB: ARTICLE
JCB823
Mutants in
endosome to the late Golgi
trs120 disrupt traffic from the early
Huaqing Cai,
1
Yueyi Zhang,
1
Marc Pypaert,
2
Lee Walker,
1
and Susan Ferro-Novick
1,2
1
T
Howard Hughes Medical Institute and
2
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06519
ransport protein particle (TRAPP), a large complex
that mediates membrane traffic, is found in two
forms (TRAPPI and -II). Both complexes share
seven subunits, whereas three subunits (Trs130p, -120p,
and -65p) are specific to TRAPPII. Previous studies have
shown that mutations in the TRAPPII-specific gene
block traffic through or from the Golgi. Surprisingly, we
report that mutations in trs120
secretion. Instead, trs120 mutants accumulate aberrant
membrane structures that resemble Berkeley bodies and
trs130
do not block general
disrupt the traffic of proteins that recycle through the
early endosome. Mutants defective in recycling also dis-
play a defect in the localization of coat protein I (COPI)
subunits, implying that Trs120p may participate in a
COPI-dependent trafficking step on the early endosomal
pathway. Furthermore, we demonstrate that Trs120p
largely colocalizes with the late Golgi marker Sec7p. Our
findings imply that Trs120p is required for vesicle traffic
from the early endosome to the late Golgi.
Introduction
The yeast
fluid, plasma membrane proteins, and ligands bound to their re-
ceptors (Riezman, 1993). The endocytosis of membrane pro-
teins from the plasma membrane to the vacuole can be divided
into several steps that involve at least two classes of endo-
somes, early and late endosomes (Singer-Kruger et al., 1993;
Hicke et al., 1997; Prescianotto-Baschong and Riezman, 1998).
The late endosome, or prevacuolar compartment, is where the
endocytic pathway converges with the
(
vps
) pathway (Davis et al., 1993; Babst et al., 1997).
Traffic from endosomes to the trans-Golgi is required for
the transport of at least three different types of cargo proteins.
Soluble vacuolar proteins, such as carboxypeptidase Y (CPY),
are examples of the first type of cargo. Vps10p, a sorting recep-
tor for CPY, forms a complex with this vacuolar protease at the
trans-Golgi and then enters vesicles targeted to a prevacuolar
compartment where Vps10p and CPY are dissociated. CPY is
transported to the vacuole, whereas Vps10p is recycled back to
the trans-Golgi via a retrograde pathway (Marcusson et al.,
1994; Cooper and Stevens, 1996). Resident proteins of the late
Golgi, which recycle between the endosome and Golgi, are ex-
amples of a second type of cargo. Well-characterized examples
Saccharomyces cerevisiae
internalizes extracellular
vacuolar protein sorting
of Golgi resident proteins are Kex2p and DPAP-A (dipeptidyl
aminopeptidase A), which act in the late Golgi to modify the
mating pheromone
?
factor as it traverses the secretory path-
way (Fuller et al., 1988). Specific signals in the cytoplasmic
portion of these proteins are required for their retrieval from
endosomes (Wilcox et al., 1992; Nothwehr et al., 1993). The
third class of proteins are integral membrane plasma membrane
proteins, such as the exocytic SNARE Snc1p. These proteins
are endocytosed from the plasma membrane and travel through
the early endosome before they reach the late Golgi, where
they are recycled back to the plasma membrane (Lewis et al.,
2000). Several proteins have been identified that regulate traf-
fic between endosomes and the late Golgi, including proteins
that are required for vesicle budding, targeting, and fusion.
In this paper, we show that Trs120p, a component of
the multiprotein complex called transport protein particle
(TRAPP) II (Sacher et al., 2001), is required for the trafficking
of proteins from the early endosome. TRAPPII is one of six
large complexes that have been implicated in the tethering of
transport vesicles to an acceptor compartment in
(for reviews see Guo et al., 2000; Whyte and Munro, 2002).
Unlike other essential subunits of the TRAPP complex, mu-
tants in
trs120
do not appear to block general secretion.
However, the
trs120-2
,
-4,
and
of GFP-Snc1p, which traffics from the early endosome to the
late Golgi. The trafficking of a second marker protein that fol-
lows a similar recycling pathway, chitin synthase III (Chs3p),
is also defective in these mutants. Interestingly, the same
S. cerevisiae
-8
mutants block the recycling
Correspondence to Susan Ferro-Novick: susan.ferronovick@yale.edu
Abbreviations used in this paper: Chs3p, chitin synthase III; COP, coat protein;
CPY, carboxypeptidase Y; PI[3]P, phosphatidylinositol-3-phosphate; TAP,
tandem affinity purification; TRAPP, transport protein particle;
protein sorting.
The online version of this article contains supplemental material.
vps , vacuolar

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JCB • VOLUME 171 • NUMBER 5 • 2005824
trs120
(COPI) complex (also called coatomer). Our findings support a
role for Trs120p in the tethering of vesicles that recycle
through the early endosome.
mutants mislocalize subunits of the coat protein I
Results
Invertase secretion is not blocked in all
trs120 temperature-sensitive mutants
Experiments have shown that there are two forms of the
TRAPP complex, TRAPPI and -II. TRAPPI and -II share seven
subunits, whereas three subunits (Trs130p, -120p, and -65p)
are unique to TRAPPII (Sacher et al., 2001). Mutational analy-
sis and in vitro transport studies have revealed that TRAPPI
acts in ER–Golgi traffic to mediate the tethering of COPII ves-
icles to the Golgi, whereas mutants in the TRAPPII-specific
subunit Trs130p disrupt Golgi traffic (Sacher et al., 2001).
To further characterize the function of TRAPPII in mem-
brane traffic, we constructed a collection of temperature-sensitive
mutations in genes that encode essential TRAPPII-specific
subunits. Initially, PCR mutagenesis was used to isolate mu-
tants in
trs120
. However, after this approach failed to yield
mutants, transposon mutagenesis was performed. The location
of the transposon insertion in each of the seven mutants we iso-
lated is shown in Fig. 1 A. An eighth mutant,
constructed by truncating the last 481 amino acids of Trs120p
(Fig. 1 A). Further deletion of the COOH terminus of Trs120p
led to cell death. Five truncation mutants in
constructed. These mutants deleted the region of
encodes the last 33–124 amino acids (Fig. 1 A). Deletion of
TRS65
, which encodes the third TRAPPII-specific subunit, did
not affect the growth of yeast or membrane traffic (Sacher et
al., 2001).
Initially, these temperature-sensitive mutants were exam-
ined for defects in the trafficking of invertase. Invertase is
translocated into the lumen of the ER, where it is core glyco-
sylated. The ER form of invertase, which is
in mutants that block traffic from the ER to the Golgi complex
(
sec18
mutant in Fig. 1, B [lane 13] and C [lane 11]). The 80-kD
form of invertase is further glycosylated in the Golgi and then
transits through the early endosome before it is secreted into
the periplasm (Harsay and Schekman, 2002). External inver-
tase migrates on SDS–polyacrylamide gels as a heterogeneous
species with an apparent molecular mass
1 B, lane 2). To determine whether mutants in
block invertase secretion, we shifted wild-type and mutant cells
to the restrictive temperature (37
block in secretion. Cells were resuspended in low-glucose me-
dium to derepress the synthesis of invertase and radiolabeled
for 60 min with [
S] ProMix. As shown in Fig. 1 B, all
mutants exhibited a near complete or partial defect in secretion
at the nonpermissive temperature, accumulating the heteroge-
neous high molecular mass form of invertase and partially gly-
cosylated forms. Invertase assays revealed that this defect was
clearly temperature sensitive (Table S1, available at http://
www.jcb.org/cgi/content/full/jcb.200505145/DC1). An analysis
of the carbohydrate modification indicated that the accumulated
trs120-1
, was
trs130
were also
TRS130
that
?
80 kD, accumulates

of 100–140 kD (Fig.
trs130
and
-120
?
C) for 20 min to impose a
35
trs130
invertase contained
Sacher et al., 2001; unpublished data), implying that it reached
the late Golgi (Graham and Emr, 1991; Brigance et al., 2000).
These results indicate that mutations in
in membrane traffic at multiple steps in the Golgi, including the
late Golgi, where invertase is fully glycosylated. In contrast, in-
vertase assays revealed partial secretion defects in only two
trs120
mutants (
trs120-2
and
-8
demonstrated that these mutants showed a partial block in se-
cretion and accumulated both partially and fully glycosylated
forms of invertase. Both mutants also secreted some external
invertase (Fig. 1 C and see Table S1 for quantitative assays).
The forms of invertase that accumulated in
as well as two mutants that did not accumulate significant
amounts of invertase, are shown in Fig. 1 C. The defect ob-
served in the
trs120-2
and
-8
mutants was reproducibly seen in
four separate experiments.
?
1-3 mannose (later Golgi modification;
trs130
confer a defect
). Radiolabeling experiments
trs120-2
and
-8
,
Figure 1.
(A) A schematic diagram of trs120 and -130 mutants used in this study.
The trs120-1 mutant was made by truncating 481 amino acids from the
COOH terminus of Trs120p. The other trs120 mutants were made by
transposon mutagenesis as described in Materials and methods. The ar-
rows point to the position where the transposon is inserted in each mutant.
Five trs130 mutants were made by truncating 33, 54, 74, 94, and 124
amino acids from the COOH terminus of Trs130p. Invertase secretion is
blocked in all trs130 mutants (B) but not all trs120 mutants (C). Wild-type
(WT) and mutant cells were grown at 25?C and preincubated at 37?C for
20 min. Cells were resuspended in low-glucose medium to induce the syn-
thesis of invertase and were radiolabeled for 60 min. Samples were then
processed for the immunoprecipitation of internal (I) and periplasmic (E)
invertase. To facilitate the detection of the high molecular mass form of
invertase in trs130 mutants, cells were transformed with the SUC2 gene
on a 2-?m plasmid.
The analysis of invertase secretion in trs130 and -120 mutants.

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EARLY ENDOSOMAL TRAFFIC • CAI ET AL. 825
The loss of
block the trafficking of CPY
To monitor the trafficking of a second marker protein, we an-
alyzed the vacuolar protease CPY. CPY is synthesized, trans-
located, and glycosylated in the lumen of the ER (p1 form)
before it is transported to the Golgi (p2), where it receives addi-
tional carbohydrate modifications. On transport to the vacuole,
CPY is proteolytically activated to the mature form (Stevens et
al., 1982). To examine CPY trafficking, wild-type and mutant
cells were preincubated at 37
?
C for 20 min, pulse labeled for 4
min, and chased for 30 min (Fig. 2 A). At the end of the chase,
all
trs130
mutants accumulated small amounts of the p1 form
of CPY as well as a heterogeneous smear of CPY that accumu-
lated between the p1 and p2 forms of CPY. Small amounts of
mature CPY were also observed at the end of the chase (Fig. 2 A,
top), suggesting that Trs130p is not completely inactivated at
37
?
C. These results are consistent with our previous proposal
that TRAPPII may be required for intra-Golgi traffic (Sacher et
al., 2001). This finding is also consistent with the observation
that fully glycosylated invertase accumulates in the late Golgi
in all
trs130
mutants. In contrast, the kinetics of anterograde
CPY trafficking in all
trs120
mutants was the same as wild
type (Fig. 2 A, bottom).
CPY is sorted away from secreted proteins in the trans-
Golgi by the sorting receptor Vps10p (Vida et al., 1993; Mar-
cusson et al., 1994). Failure to sort CPY from the TGN or to
retrieve Vps10p from a late endosomal compartment leads to a
vps
phenotype and the secretion of CPY into the growth me-
dium (Bankaitis et al., 1986; Rothman and Stevens, 1986).
trs120 function does not
To determine whether the loss of
to a
vps
phenotype, wild-type and mutant cells were grown to
early log phase, pelleted, resuspended in fresh growth medium,
and shifted to 37
?
C for 2 h. Proteins secreted into the growth
medium were precipitated with TCA and assayed for the pres-
ence of CPY using Western blot analysis. Although the secre-
tion of CPY was detected in the
top [lane 7] and bottom [lane 10]), significant amounts of CPY
were not detected in the
trs120
and
trs120
or
-130
function leads
vps
mutant,
vps1
?
(Fig. 2 B,
-130
mutants (Fig. 2 B).
Trs120p is not required for traffic on
the exocytic pathway
The finding that
trs120-2
and
ficking of CPY and only partially block invertase secretion
prompted us to determine whether there is a general block in
secretion in these mutants. To assay for a general secretion
defect, we shifted wild-type and mutant cells to 37
min, pulse labeled them for 15 min, and chased them for 30
min. Cells were pelleted, and the proteins secreted into the
medium were precipitated with TCA and resolved on an
SDS–polyacrylamide gel (Fig. 3). Surprisingly, although the
sec18
,
trs130
, and
trs130-6
(Fig. 3, compare lane 1 with lanes 2, 5, and 6), no obvious de-
fect was observed in the
trs120-2
The block in secretion was complete in
trs130
mutants (see Fig. S1, available at http://www.jcb.org/
cgi/content/full/jcb.200505145/DC1, for darker exposure of
the autorad). Therefore, although mutations in the
lead to a defect in the trafficking of proteins within the Golgi,
the loss of
trs120
function does not result in a significant
block in secretion.
-8
mutants do not block the traf-
?
C for 20
ts2
mutants blocked secretion
and
-8
mutants (lanes 3 and 4).
sec18
but not in
trs130
gene
Figure 2.
(A) Mutants in trs130, but not -120, display a defect in CPY trafficking.
Wild-type (WT) and mutant cells were grown at 25?C, preincubated at
37?C for 20 min, labeled for 4 min, and chased for 30 min. Aliquots of
samples were removed at the indicated time points and immunoprecipi-
tated with anti-CPY antibody. (B) Mutants in trs130 and -120 do not se-
crete CPY into the growth medium. The growth medium was collected from
wild-type and mutant cells that were shifted to 37?C for 2 h, processed for
TCA precipitation, and blotted with anti-CPY antibody. The secretion of
CPY from the vps1? mutant is shown as a control.
The analysis of CPY trafficking in trs130 and -120 mutants.
Figure 3.
grown to early log phase at 25?C, preincubated to 37?C for 20 min, radio-
labeled for 15 min, and then chased for 30 min. The growth medium was
separated from the cells by centrifugation, processed for TCA precipita-
tion, and analyzed by SDS-PAGE on a 10% gel as described previously
(Kim et al., 2001). The asterisks mark proteins secreted into the medium in
wild-type (WT) cells.
Mutants in trs120 do not block general secretion. Cells were

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JCB • VOLUME 171 • NUMBER 5 • 2005826
The recycling of Snc1p and Chs3p is
defective in trs120 mutants
Our findings indicate that Trs120p may not be required for
general secretion. However, the analysis of invertase traffick-
ing revealed a defect in invertase secretion in the trs120-2 and -8
mutants (Fig. 1 C and Table S1). Invertase has been shown to
transit through the early endosome before reaching the cell sur-
face (Harsay and Schekman, 2002). Therefore, one explanation
for the defect in invertase secretion in the trs120-2 and -8 mu-
tants is that Trs120p may be required for the trafficking of pro-
teins through the early endosome. To begin to address this
question, we examined the recycling of GFP-Snc1p in the
trs120 mutants we constructed, as well as in all trs130 mutants.
Snc1p is the yeast homologue of synaptobrevin. With its
homologous partner, Snc2p, it mediates the fusion of post-
Golgi vesicles with the plasma membrane (Protopopov et al.,
1993). Snc1p largely resides on the plasma membrane but is
rapidly endocytosed and traffics through the early endosome
before it reaches the late Golgi, where it is incorporated into
another round of secretory vesicles (Fig. 4 A; Lewis et al.,
2000). To monitor the recycling of GFP-Snc1p, we grew wild-
type and mutant cells at the permissive temperature (25?C) or
shifted them to 37?C for 60 min. In wild-type cells, GFP-Snc1p
was generally found at regions of polarized growth and the
plasma membrane. Interestingly, after a 60-min shift to 37?C,
little GFP-Snc1p was found at the cell surface in the trs120-2
and -8 mutants (Fig. 4 B). In both mutants, GFP-Snc1p was
present on intracellular membranes in ?80% of the cells. In
trs120-2, GFP-Snc1p was found in small punctate structures at
25 and 37?C. The recycling defect in trs120-2 resulted in a
growth defect at 25?C, and when this defect was more pro-
nounced at 37?C, the cells died. An increased cytoplasmic haze
was also observed (Fig. 4 B). This cytosolic haze may corre-
spond to the presence of GFP-Snc1p in transport vesicles. In
support of this hypothesis, we found that the trs120-2 mutant
accumulated three to five times more vesicles than wild type
(see Fig. 9). Although no significant defect in invertase secre-
tion was found in the trs120-4 mutant, GFP-Snc1p was found
in punctate structures in ?40% of the cells (Fig. 4 B). Thus,
trs120 mutants that exhibited defects in invertase secretion also
displayed severe defects in GFP-Snc1p recycling. In contrast,
all trs130 mutants were defective in GFP-Snc1p recycling and,
like trs120-8, GFP-Snc1p accumulated in larger structures in
these mutants (Fig. 4 B, trs130ts2).
As shown in Fig. 4 C, GFP-Snc1p–containing structures
that accumulate in trs120 and -130 mutants became larger dur-
ing a 60-min incubation at 37?C. To address whether the en-
larged structures are early endosomes or Golgi, we labeled
trs130ts2and -120-8 mutant cells that were shifted to 37?C for
30 min with the styryl dye FM4-64. The early endosome is la-
beled after a brief incubation (5 min) with this dye (Vida and
Emr, 1995). At the 5-min time point, the GFP-Snc1p–containing
structures were labeled with FM4-64 (Fig. 5), implying that at
least some of the Snc1p accumulates in the early endosome at
Figure 4.
GFP-Snc1p recycling. (A) The recycling pathway of GFP-
Snc1p. (B) GFP-Snc1p was examined in wild-type (WT)
and mutant cells that were grown at 25?C (0 min) or
shifted to 37?C for 60 min. (C) GFP-Snc1p was examined
in wild type, trs130ts2, and trs120-8 mutants at 0, 5, 15,
30, and 60 min after a shift to 37?C.
Mutants in trs120 and -130 are defective in

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EARLY ENDOSOMAL TRAFFIC • CAI ET AL. 827
37?C in the trs130ts2 and -120-8 mutants. At the later time point
(30 min), FM4-64 was no longer detected in the GFP-Snc1p–
containing structures and instead stained the vacuolar mem-
brane, which was fragmented in both mutants (Fig. 5).
To determine whether another marker protein that travels
through the early endosome is also defective in these trs120
and -130 mutants, we analyzed the cell surface enzyme Chs3p.
Chs3p follows a recycling pathway that is similar to Snc1p.
It localizes to the mother-bud junction, the cell surface, and
punctate intracellular structures called chitosomes (Chuang and
Schekman, 1996; Valdivia et al., 2002). To monitor the recy-
cling of Chs3p, we grew wild-type and mutant cells at the
permissive temperature or shifted them to 37?C for 30 min.
Quantitation of ?100–150 cells revealed that at 37?C Chs3p-
GFP localized to the mother–bud neck region in ?51% of
wild-type cells. A dramatic decrease in the targeting of Chs3p-
GFP to the bud neck was observed in the trs130ts2 (8%), -120-2
(10.5%), and -120-8 (9%) mutants at this temperature, whereas
a modest decrease was seen in the trs120-4 (29%) mutant (Fig. 6).
The trs120-2 mutant also displayed a partial defect (30%) at
25?C, whereas no defect was observed in the trs130ts2, -120-4,
and -120-8 mutants at this temperature (Fig. 6). These findings
indicate that mutants in trs120 and -130 display defects in the
targeting of Chs3p to the bud neck. Furthermore, the tempera-
ture dependence and the severity of the defect for each of the
affected mutants was the same as observed for GFP-Snc1p.
TRAPPII localizes to a late Golgi/early
endosomal compartment
Previous fractionation studies indicated that TRAPPI and -II
cofractionate with each other and early Golgi markers on a su-
crose density gradient (Sacher et al., 2001). Our finding that mu-
tations in trs130 and -120 block the trafficking of Snc1p and
Chs3p warranted a reexamination of the localization of TRAPPII.
In an attempt to better resolve the different compartments
of the Golgi by subcellular fractionation, we modified the sucrose
density gradient we had previously used. In earlier studies, a
step gradient containing 11 different concentrations of sucrose,
ranging from 18 to 60%, was used. The TRAPPII complex
fractionated between 26 and 48% sucrose on this gradient
(Sacher et al., 2001). For this paper, we designed a new step
gradient containing 11 1-ml steps that ranged from 26 to 50%
sucrose (see details in Materials and methods). Och1p, which
marks the earliest known carbohydrate-modifying compart-
ment in the yeast Golgi, was used as an early Golgi marker
(Nakayama et al., 1992; Brigance et al., 2000), whereas Chs3p
marked both the late Golgi and early endosomes (Valdivia et
al., 2002). Late Golgi and early endosomes are difficult to
resolve by sucrose density analysis (Holthuis et al., 1998;
Valdivia et al., 2002). Thus, it is unclear whether late Golgi
markers solely mark this compartment or if they also mark the
early endosome. With our gradient conditions, Och1p peaked
in fraction 4 (Fig. 7 A), whereas Chs3p peaked in fraction 8
(Fig. 7 C). The SNARE Sec22p peaked in fractions 3 and 11
(Fig. 7 B). The later peak (at 47% sucrose) represents the frac-
tion of Sec22p that resides on the ER, whereas the earlier peak
(at 29% sucrose) is Golgi-bound Sec22p (Barrowman et al.,
2000). This result confirms that our new gradient conditions
clearly resolve early and late Golgi subcompartments. To mon-
itor the localization of TRAPPII on this gradient, we added a
13-x-myc tag to the genomic copy of Trs120p as described pre-
viously (Longtine et al., 1998). Tagged Trs120p is functional,
as the presence of the tag did not impede growth or the assem-
bly of the TRAPPII complex. Trs120p largely cofractionated
with Chs3p (Fig. 7 C) but not with Och1p and Sec22p (Fig. 7,
Figure 5.
-120-8 mutants. Cells were labeled with FM4-64 as described in Materials
and methods. FM4-64 was internalized for 5 or 30 min.
GFP-Snc1p localizes to early endosomes in the trs130ts2 and
Figure 6.
Chs3p-GFP. Chs3p-GFP was examined in wild-type and mutant cells that
were grown at 25?C or shifted to 37?C for 30 min. Arrows point to bud
necks and incipient bud sites, which are visible in the differential interfer-
ence contrast (DIC) image.
Mutants in trs120 and -130 are defective in the trafficking of