Department of Biochemistry and Molecular Biology, Louisiana State
University Health Sciences Center, Shreveport, LA 71130, USA
* Present address: SUNY-Buffalo, Department of Biochemistry, 140 Farber Hall,
Buffalo, NY 14214, USA
Author for correspondence (e-mail:
lrobin{at}lsuhsc.edu)
Accepted 27 September 2002
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Summary |
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Key words: Casein kinase 1, GFP fusion proteins, Protein targeting, AKR1
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Introduction |
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The budding yeast Saccharomyces cerevisiae encodes four CK1
isoforms: Yck1p, Yck2p, Yck3p and Hrr25p
(DeMaggio et al., 1992;
Hoekstra et al., 1991
;
Robinson et al., 1992
;
Wang et al., 1992
;
Wang et al., 1996
). These four
enzymes are strongly conserved with their higher eukaryotic counterparts,
exhibiting greater than 50% amino acid identity through their catalytic
domains. Hrr25p is a nuclear protein that is important for DNA replication and
repair (DeMaggio et al., 1992
;
Hoekstra et al., 1991
). Yck3p
shares some function with Hrr25, as deletion of YCK3 in an
hrr25 genetic background is lethal
(Wang et al., 1996
). However,
Yck3p is distributed throughout the cell
(Wang et al., 1996
).
YCK1 and YCK2 form a functionally redundant gene pair that
is essential for viability (Robinson et
al., 1992
; Wang et al.,
1992
). Deletion of either gene does not strongly affect cells, but
deletion of both genes causes aberrant cellular morphology and growth arrest
(Robinson et al., 1992
;
Wang et al., 1992
).
The Yck1p and Yck2p protein kinases are involved in numerous cellular
processes, including bud morphogenesis
(Robinson et al., 1993),
internalization of plasma membrane permeases
(Marchal et al., 2000
) and
pheromone receptors (Hicke et al.,
1998
; Panek et al.,
1997
), and cytokinesis
(Robinson et al., 1993
). Yck2p
is a 62 kDa protein that is tightly associated with the plasma membrane
(Vancura et al., 1994
), and
biological function depends on membrane association. CK1 isoforms generally
comprise an N-terminal kinase domain and a highly divergent C-terminal domain
that often is responsible for localization
(Gross and Anderson, 1998
).
Subcellular localization is necessary and sufficient for defining the
functions of the yeast isoforms (Wang et
al., 1996
). For example, the nuclear isoform Hrr25p complemented
yck1 yck2 mutant strains when Hrr25p was engineered to contain the
Yck2p -Cys-Cys site at its C-terminus
(Wang et al., 1996
).
Conversely, a chimeric protein comprised of the Yck2p catalytic domain and an
Hrr25p region, including its nuclear localization signal, complements an
hrr25 null mutant (Vancura et
al., 1994
).
Although Yck1p and Yck2p C-termini share very little overall sequence
identity, both contain Gln-rich sequences
(Robinson et al., 1992;
Wang et al., 1992
) and a 12
residue C-terminal sequence with 83% sequence identity that terminates with
the sequence -Cys-Cys. The two C-terminal Cys residues are essential for Yck2p
membrane association and function
(Robinson et al., 1993
;
Vancura et al., 1993
;
Vancura et al., 1994
).
Experiments to determine the nature of Yck2p membrane association demonstrated
that Yck2p is solubilized only by a combination of salt and nonionic detergent
or the use of sodium dodecyl sulfate
(Vancura et al., 1993
). These
findings, in conjunction with the presence of the Cys-Cys motif, led to the
proposal that Yck2p is tethered to the inner leaflet of the plasma membrane
via prenylation (Vancura et al.,
1993
). However, the enzymology of the relevant enzyme argues
against this possibility (Desnoyers et
al., 1996
), and it was reported recently that Yck2p localization
requires the Akr1 protein (Feng and Davis,
2000
) and that Yck2p is a substrate for palmitoyl transferase
activity of Akr1p (Roth et al.,
2002
). It is likely that both terminal Cys residues are modified
in this way.
We have been interested in how Yck2p is targeted specifically to the plasma
membrane. Palmitoylated proteins with no other lipid modification are
generally plasma membrane associated, but there are few examples where the
targeting mechanism is understood. Palmitoyl transferase activities have been
observed in plasma membrane fractions and to a lesser degree in Golgi
membranes (Resh, 1999).
However, only recently have proteins with palmitoyl transferase activity been
identified, and their distributions within the cell are often at internal
membranes (Lobo et al., 2002
;
Roth et al., 2002
). Two
potential mechanisms of plasma membrane association of palmitoylated proteins
are that they first associate with the plasma membrane and become modified
there, or that they are modified at an internal membrane and then targeted to
the plasma membrane. The SNAP-25 protein, for example, is thought to utilize
the secretory pathway for targeting
(Gonzalo and Linder, 1998
;
Loranger and Linder, 2002
).
The palmitoyl moiety itself could provide a plasma membrane targeting signal,
since palmitoylation-deficient Ras proteins fail to move from internal
membranes to the plasma membrane (Apolloni
et al., 2000
; Choy et al.,
1999
).
We previously reported that Yck2p is differentially enriched at sites of
polarized secretion during the cell cycle
(Robinson et al., 1999). The
localization pattern we observed resembles that of proteins directed to the
plasma membrane via the classical secretory pathway. Here, we report that
secretory function, including ER-Golgi trafficking, is necessary for Yck2p
plasma membrane localization. Furthermore, we show that the two terminal Cys
residues are necessary but insufficient for proper Yck2p targeting; C-terminal
sequences upstream of the Cys residues are required to direct GFP-Yck2p plasma
membrane targeting.
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Materials and Methods |
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DNA manipulation
E. coli strains DH5 and XL1blue were used for plasmid
amplification and subcloning. Restriction enzymes (Promega; American Allied
Biochemicals), Klenow fragment of DNA polymerase 1 (Promega) and DNA ligase
(New England Biolabs) were used according to manufacturer's recommendations.
Plasmid DNA was purified either by an alkali lysis method or by a rapid
boiling preparation (Taylor et al.,
1993
). For DNA sequence analysis, either preparation was further
purified by RNase treatment followed by precipitation from polyethylene glycol
8000. PCR amplification was carried out with Bio-X-Act polymerase (Bioline)
using a Perkin-Elmer 9600 or a GeneAmp 2400 (Applied Biosystems) thermocycler.
DNA sequence analysis of cloned PCR products and of mutagenesis products was
carried out either manually, using the Sequenase (US Biochemical) dideoxy
chain termination method, or by automated sequencing (Iowa State University
DNA Sequencing and Synthesis Facility or Retrogen).
Construction of plasmids and mutant alleles
Plasmids used for this work are listed in
Table 2. High copy plasmid
pL2.35 (YEp352:GFP:YCK2) was constructed by cloning the
XbaI-SacI fragment, containing the GFP:YCK2 fusion
gene with flanking sequences, from pL2.991 [pUCEco:GFP:YCK2
(Robinson et al., 1999
)] into
YEp352 (2µ, URA3).
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The pJB9 plasmid was constructed to allow expression of YCK variants from the GAL1 promoter. An EcoRI-BamHI GAL1 promoter fragment was digested from plasmid pGal-CLB5 (kindly provided by C. Wittenberg) and cloned directly into YCp50. YCK variant open reading frames (ORFs) were generated with 5' BamHI and 3' SalI sites, and these sites were used for cloning all YCK2 ORF variants into pJB9.
The pPB23 plasmid was constructed as a cassette vector to place any
GFP:YCK2 variant ORF in the context of natural YCK2 flanking
sequences. The SalI site of pUC19 was destroyed by SalI
digestion followed by a fill-in reaction with Klenow fragment, yielding
pUC19Sal. The YCK2 gene with flanking sequences was cloned
into pUC19
Sal on an XbaI-SacI fragment, yielding
plasmid pPB18. A BamHI site was introduced into pPB18 following the
YCK2 ATG codon by inverse PCR with primers PB13 and PB14
(Table 2), yielding plasmid
pPB19. A SalI site was also introduced into pPB18 following the
YCK2 stop codon by inverse PCR using primers PB15 and PB16
(Table 2), yielding plasmid
pPB21. Finally, the pPB21 HindIII-SacI fragment with the
introduced SalI site was swapped into pPB19, yielding plasmid
pPB23.
The yck22-360 allele encodes residues 361 to 546, lacking
all catalytic domain-encoding sequences. This allele was generated by PCR
using primers Y2CORF1A and Y2ORF2X (Table
3) on template pL2.99. The product was cloned into pUC
Eco
(Robinson et al., 1999
) to
yield pL210. After correct YCK2 sequence was confirmed, the F64L S65T
GFP allele was cloned into the EcoRI site following the initiating
ATG of yck2
2-360, yielding plasmid pL211. The GFP fusion gene
was then cloned into pJB9 for expression from the GAL1 promoter,
yielding plasmid pL212. The BamHI-SalI fragment was also
swapped into pPB23 to place GFP:yck2
2-360 under control of the
YCK2 promoter, yielding plasmid pL221. The
XbaI-SacI fragment containing the fusion allele was cloned
into the low copy vector pRS316, yielding plasmid pL222.
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The yck2397-532 mutant allele, lacking all but the final 14
residues of the C-terminal domain (CTD), was constructed by inverse PCR.
Primers YCK-CG1 and YCK-CG4 (Table
3) were designed to add HindIII sites after codons 396
and 532, respectively. PCR with these primers on template pJB4-4
(GFP:YCK2 in pUC
Eco) resulted in a linear product with
terminal HindIII sites lacking codons 397 to 532. This product was
digested with HindIII and religated to yield plasmid pL230. The
entire yck2
397-532 allele was digested from this plasmid with
XbaI and SacI and cloned into pRS316 to yield plasmid
pSJ23.
Mutant alleles encoding C-terminal deletions were constructed using the
QuikChange mutagenesis kit (Stratagene) with pJB4-4 (GFP:YCK2 ORF
fusion in pUCEco) as template. Primers are listed in
Table 3. In each case, the
mutant GFP:YCK2 ORF was cloned into pJB9 for expression from the
GAL1 promoter. For expression from the YCK2 promoter, each
mutant ORF was cloned into pPB23. The entire allele then was cloned on an
XbaI-SacI fragment into pRS316. The GFP:YCK2-CIIS
ORF was generated by PCR using primers Y2ORF1G1 and Y2CAAX2
(Table 3) on template pJB4-4.
The PCR product was digested and the resulting fragment was ligated into
pUC19
Eco. Among products with correct sequence was plasmid pL250. The
YCK2 ORF was excised from pL250 and cloned into pJB9 for expression
from the GAL1 promoter, yielding plasmid pL251.
To construct the YCK2/YCK1 chimera, two separate PCR products were
generated that encode GFP fused to the Yck2p catalytic domain and the Yck1p
C-terminal domain. The YCK2 product, with BamHI site at the
5' end and SphI site at the 3' end, was generated on
template pLR10 (GFP:YCK2 in pUC19Sal) with primers Y2ORF1G1
and Y2/1-mid5'. The YCK1 product, with SphI site at
the 5' end and SalI site at the 3' end, was generated
using primers Y2/1-mid3' and Y2/1-3' with pLJ721 [YCK1 in
YEp352 (Robinson et al.,
1992
)] as template. Purified products were digested with
BamHI and SphI (YCK2 fragment) or SphI and
SalI (YCK1 fragment). The resulting fragments were ligated
together in the presence of BamHI and SalI-digested pJB9.
Ligation products with intact YCK2 and YCK1 sequences
included pL240. For expression from the YCK2 promoter, the GFP fusion
ORF was swapped into pPB23, yielding pL241. The XbaI-SacI
fragment containing the chimera with YCK2 flanking sequences was
cloned into pRS316, yielding plasmid pL242.
Protein analysis
For induction of GFP fusion expression from the GAL1 promoter,
cells were first grown in synthetic media containing 2% raffinose as carbon
source. Galactose was then added to cultures to a final concentration of 2%,
and cultures were incubated at indicated temperature, removing samples for
microscopic observation and/or for protein isolation at times indicated.
For immunoblot analysis, total protein extracts were prepared by TCA
precipitation (Davis et al.,
1993) from cells grown to OD at 600 nm of 0.5-0.9. Protein
extracts were separated by electrophoresis through pre-cast denaturing
SDS-polyacrylamide gels (Bio-Rad and Fisher Scientific). Gels were blotted to
nitrocellulose and blots were probed with affinity-purified antiserum against
GFP (kindly provided by J. N. Davis, Louisiana State University Health
Sciences Center, Shreveport). Horseradish peroxidase-conjugated secondary
antisera (Sigma) and the ECL chemiluminescence kit (Amersham) were used to
detect the primary antibody. To control for loading, blots were stripped and
reprobed for phosphoglycerate kinase (PGK), using a monoclonal antibody
(Molecular Probes).
Fluorescence microscopy
Microscopy to examine GFP fluorescence in live cells was performed using an
Olympus AX70 Provis microscope equipped for differential interference contrast
optics and epifluorescence, as described previously
(Robinson et al., 1999).
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Results |
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Induction of GFP-Yck2p synthesis in a wild-type strain was monitored by immunoblot analysis and fluorescence microscopy at intervals after addition of galactose. As shown in the immunoblot in Fig. 1A, the fusion protein is below detection level in cells grown on glucose (left panel, lane 1) and on the derepressing carbon source raffinose (t=0 minutes; right panel). Following addition of galactose to derepressed cultures, GFP-Yck2p is detectable at low levels after 30 minutes; after 90 minutes the level of fusion protein is comparable with the steady-state level in cells expressing the fusion protein from the YCK2 promoter at its endogenous chromosomal locus (Fig. 1A, compare lane 2, left panel, with 90 minutes lane in right panel). Further induction results in levels comparable with those in cells expressing the fusion protein from a high copy (2µ) plasmid (Fig. 1A, compare lane 3 with the 120 minutes time point).
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Microscopic observation of GFP-Yck2p induction revealed a similar time course. Fluorescent signal detectable above background was observed first at 30 minutes of induction (Fig. 1B). At time points up to 60 minutes, GFP fluorescence was mainly cytosolic and punctate, but was brightest at sites of polarized growth, below the membrane of small buds and the bud necks of dividing cells. At 60 minutes, the plasma membrane was labeled in 40-50% of the cells (n=200). At 90 minutes of induction, cells resembled those expressing the fusion from the YCK2 promoter at steady state. All detectable fluorescence was plasma membrane associated and was enriched at small bud membranes and at the necks of dividing cells. These results are consistent with the idea that Yck2 protein associates with internal membranes early after synthesis and remains associated with membrane-limited compartments during targeting to the plasma membrane.
To test directly whether vesicle-mediated secretory pathway function is
required for GFP-Yck2p targeting to the plasma membrane, we introduced the
pGal:GFP:YCK2 plasmid (pJB1) into each of eight conditional
secretory pathway-defective [sec
(Novick et al., 1980)]
mutants. These temperature-sensitive (ts) mutations block secretory traffic at
discrete steps upon shift to restrictive temperature but protein synthesis
continues, resulting in accumulation of protein at the affected compartment.
The sec mutants used here cause vesicle trafficking blocks at the
following steps: vesicle budding from the ER [sec12, sec23
(Barlowe et al., 1994
)];
consumption of ER-derived vesicles at the Golgi [as well as fusion of vesicles
at all subsequent steps; sec18
(Eakle et al., 1988
)];
trafficking through the Golgi [sec14
(Bankaitis et al., 1989
)]; and
docking and fusion of secretory vesicles with the plasma membrane
[sec1 (Egerton et al.,
1993
); sec4, sec8
(TerBush and Novick, 1995
);
and sec9 (Brennwald et al.,
1994
)]. The location of fluorescence was examined after induction
of GFP-Yck2p synthesis at permissive and restrictive temperature in each
mutant.
Induction of GFP-Yck2p synthesis in each mutant at permissive temperature
resulted in plasma membrane localization
(Fig. 2A shows sec18,
sec23, sec14, sec4 and sec9). However, after 90 minutes of
induction at the restrictive temperature, cells of each of the sec
mutants showed only punctate intracellular fluorescence. The internal
fluorescence patterns at restrictive temperature were generally consistent
with patterns predicted by the nature of the blocks, although sec23
cells did not always show perinuclear fluorescence consistent with ER
accumulation. Perinuclear fluorescence was observed in fewer than 20% of
sec23 cells, with the majority of cells showing bright dots. This
pattern is more reminiscent of endosomal labeling than of ER structures. Since
the length of the shift to restrictive temperature is relatively long, it was
possible that we were not observing a primary result of the sec23
block in these cells, but instead, a secondary effect on recycling from the
plasma membrane, as documented in this mutant for the plasma membrane SNARE
protein Snc1p (Lewis et al.,
2000). If this were the case, it would suggest that Yck2p is
modified at the plasma membrane but is rapidly recycled, and that our
sec blocks revealed only a block to this recycling.
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To test this possibility, we performed an identical shift with a strain
mutant for both sec23 and end4. Endocytosis is blocked in
the end4-1 mutant at 37°C
(Raths et al., 1993), which is
also the restrictive temperature for sec23-1. If the Yck2 protein is
associated with early secretory membranes after synthesis rather than with
endosomal or Golgi membranes after internalization from the plasma membrane,
we expected that we should observe a pattern identical to that observed for
cells of the single sec23 mutant. By contrast, if recycling results
in the pattern observed for the single sec23 mutant, we expected to
observe plasma membrane accumulation in the double sec23 end4 mutant.
As shown in Fig. 2B, double
mutant cells appear identical to single sec23 mutant cells at both 24
and 37°C, indicating that the block to ER exit likely results in
accumulation of GFP-Yck2p on an early secretory membrane. Perinuclear
fluorescence may not be observed often in these cells at least partly as a
result of changes to organization of early secretory membranes that occur
after a prolonged shift to restrictive temperature
(Lewis et al., 2000
).
Hydrophobic modification of Yck2p is required for secretory membrane
association
Plasma membrane association and biological function of Yck2p and GFP-Yck2p
require the two terminal Cys residues
(Robinson et al., 1999;
Robinson et al., 1993
;
Vancura et al., 1994
). There
is now strong evidence that the modification on one or both Cys residues is
palmitate, with addition catalyzed by the Akr1 protein
(Roth et al., 2002
). By two
hybrid analysis, Akr1p has been reported to interact not only with plasma
membrane proteins of the mating signal transduction pathway
(Givan and Sprague, 1997
;
Kao et al., 1996
;
Pryciak and Hartwell, 1996
),
but also with Gcs1p (Kao et al.,
1996
), which functions at ER and/or Golgi membranes
(Poon et al., 1999
). Thus,
Yck2p could be modified at a secretory membrane, or could associate with a
targeting protein in an unmodified state and transit to the plasma membrane
for modification.
Preliminary immunofluorescence analysis of a tagged Akr1p suggested
location on intracellular membranes (Roth
et al., 2002), but the identity of the membrane(s) is not yet
clear. If Yck2p requires modification before association with secretory
membranes, then no association of a Yck2-Cys545,546Ser mutant
protein should occur. To test this, we expressed the
GFP-Yck2-Cys545,546Ser fusion protein
(Fig. 3) in early
(sec23) and late (sec9) acting sec mutants. In
neither case was the GFP-Yck2-Cys545,546Ser protein obviously
associated with intracellular structures at permissive or restrictive
temperature (Fig. 4;
GFP-Yck2-Cys545,546Ser panels). Thus, interaction of Yck2p with
secretory membranes requires the C-terminal Cys residues, which are probably
modified before targeting through the pathway.
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As mentioned previously, Yck2p is probably modified by Akr1-mediated
palmitoylation. Palmitoylation affects protein-protein interactions between a
number of partners (Dunphy and Linder,
1998; Resh, 1999
),
so Yck2p targeting could require palmitoylation at the C-terminus. We tested
whether a CAAX signal sequence that directs addition of a single farnesyl
group by the farnesyl transferase can substitute for the -Cys-Cys sequence. We
used -Cys-Ile-Ile-Ser, the Ras2p terminal sequence, to direct farnesyl
modification. The GFP-Yck2-CIISp protein
(Fig. 3) provides Yck function
when expressed from the Gal promoter, as indicated by complementation of the
temperature-conditional yckts mutant (yck1-
1
yck2-2ts; Fig.
5A). Although there is some vacuolar membrane fluorescence,
probably due to overexpression, the GFP-Yck2-CIIS protein localizes primarily
to the plasma membrane (Fig. 4,
GFP-Yck2-CIIS 24°C panels), consistent with the complementation data.
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To confirm that modification of the Yck2-CIIS variant differed from that of wild-type Yck2p, GFP-Yck2-CIISp was expressed in strain LZY103, which is deleted for AKR1. While GFP-Yck2p is not detectably present at any membrane in this strain, GFP-Yck2-CIISp is plasma membrane associated in LZY103 cells, as it is in wild-type cells (Fig. 6). These results confirm that the Yck2-CIIS protein does not require Akr1p function for membrane association.
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Interestingly, plasma membrane localization of the GFP-Yck2-CIIS fusion protein is blocked in secretory mutants at restrictive temperature (37°C; sec23 and sec9 cells are shown in Fig. 4), indicating that this protein can associate with secretory membranes and probably follows a similar trafficking route to wild-type Yck2p. These results show that different C-terminal modification does not abolish plasma membrane targeting of Yck2p via the secretory pathway, and further indicate that Yck2p sequences in addition to the two Cys residues determine the targeting route.
The Yck2p C-terminal third is necessary and sufficient for
modification and targeting
To determine which Yck2p sequences are important for modification and for
targeting via the secretory pathway, we carried out deletion analysis. We
first expressed an N-terminally truncated GFP-Yck2 fusion protein that lacks
the entire catalytic domain but retains the wild-type C-terminal 185 residues
(GFP-yck22-360; Fig. 3).
It was reported previously that replacing the C-terminal sequences of Hrr25
with those of Yck2p resulted in a fusion protein that could function as Yck2p
(Wang et al., 1996
). As
predicted by these results, the GFP-yck2
2-360 fusion protein is
targeted to the plasma membrane (Fig.
4, GFP-yck2
2-360, 24°C panels). As observed for the
full-length Yck2 protein, targeting of the truncated yck2
2-360 protein
requires secretory pathway function. Both sec9 and sec23
cells incubated at restrictive temperature show only internal fluorescence for
this mutant (Fig. 4,
GFP-yck2
2-360, 37°C panels). These results demonstrate that the
sequences sufficient for secretory membrane association and plasma membrane
targeting are contained within the C-terminal third of the Yck2 protein.
To determine whether the entire 185 amino acid C-terminal sequence is
required for targeting, we deleted 137 amino acids beyond the catalytic
domain, moving up the final 14 amino acids to the end of the catalytic domain
(yck2397-532; Fig.
3). The final 14 amino acids were left because they include the
two Cys residues and also residues shared with Yck1p. The resulting GFP fusion
protein fails to complement the yckts conditional mutant
(Fig. 5B), and the majority of
its fluorescence is not associated with the plasma membrane. As shown in
Fig. 7, fluorescence from this
fusion protein was generally soluble signal. Thus, residues within the
C-terminus, in addition to the terminal Cys-Cys sequence and the 12 residues
upstream, are probably necessary for modification.
|
Sequences proximal to the terminal Cys residues are required for
modification and targeting
Aside from Gln-rich sequences, the C-terminal domain of Yck2p shares no
obvious sequence similarity with known proteins, and the S. pombe
homolog Cki1p was crystallized without this domain
(Carmel et al., 1994;
Xu et al., 1995
). Therefore,
we made a series of consecutive deletions throughout the C-terminal 185
residues to uncover sequences important for modification and/or localization
(Fig. 3). We deleted a sequence
shared with Yck1p (yck2
374-390), a sequence that includes a
group of four consecutive His residues (yck2
391-412), the four
His residues separately (yck2
406-409), the two Gln-rich tracts
(yck2
413-444, and yck2
471-498), the sequences
between these Gln-rich tracts (yck2
445-470), a sequence that
could form an
-helix (yck2
519-527), and two additional
regions close to the terminal Cys residues (yck2
499-518 and
yck2
528-540).
Each deletion allele was expressed as a GFP fusion from the GAL1 and YCK2 promoters in wild-type and yckts strains, and products were assayed for localization and for complementation of loss of Yck activity, respectively. We expected one of three outcomes for effect on localization: no effect, indicating that the sequence lost is dispensable for modification and targeting; increased cytosolic fluorescence, suggesting that the deleted sequence is important or necessary for modification; or accumulation on internal membranes, suggesting that the deleted sequence is important for targeting to secretory membranes. The latter two outcomes are not mutually exclusive, so we expected that mutants could exhibit both defects, which could indicate roles for such sequences in both modification and targeting. To classify the mutants, all were compared to the GFP-Yck2Cys545,546Ser mutant protein and to GFP-Yck2p trapped internally by imposition of a sec block.
None of the deletions closest to the catalytic domain, including the region
shared with Yck1p and the two Gln-rich tracts, affected membrane association,
plasma membrane targeting or biological function (L.C.R., R.T.C., B.M.F. and
P.B., unpublished). Thus, these sequences are dispensable for modification and
targeting. However, the three deletions removing sequences closest to the
terminal Cys residues, yck2499-518, yck2
519-527 and
yck2
528-540, each impaired plasma membrane association. The
most upstream of these sequences appears important for both modification and
targeting (Fig. 7). Cells from
log phase cultures expressing GFP-yck2
499-518p showed mainly soluble
and particulate internal fluorescence, but some fluorescence was observed at
the plasma membrane. This mutant allows growth of the
yckts strain at restrictive temperature
(Fig. 5B), possibly due to
accumulation of the mutant protein at the plasma membrane over time. Up to 50%
of cells taken from overnight plate cultures showed approximately equal levels
of plasma membrane and internal fluorescence (L.C.R., unpublished).
The nine codon deletion in yck2519-527 had the most
dramatic effect on the resulting protein. This sequence appears to be
essential for modification (as demonstrated by complete lack of visible
membrane-associated fluorescence; Fig.
7), and the mutant fails to complement the temperature-sensitive
(ts) growth of the yckts strain
(Fig. 5B). However, the
sequence is essential for accumulation of Yck2 protein. The
GFP-yck2
519-527 protein was detected at low levels by fluorescence
microscopy. We confirmed the low level of protein relative to wild-type
GFP-Yck2p or other C-terminal deletion mutants by immunoblot
(Fig. 8). Since the gene is
expressed from the GAL1 promoter, we attempted to determine the
half-life of the mutant protein by promoter shut-off experiments, but the
mutant protein failed to accumulate to reasonable levels during the pulse.
Thus, either the message or the protein is unstable. Therefore, although the
fluorescence pattern of GFP-yck2-
519-527p suggests loss of
modification, the lack of protein accumulation compromises clear
interpretation of the results.
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Deletion of a 13 amino acid sequence close to the C-terminus
(yck2528-540p) gave results consistent with loss of sequences important
for both targeting and modification. When expressed from YCK2
sequences carried in a low copy plasmid, GFP-yck2
528-540p fails to
complement the yckts mutant for ts growth or for
morphology at restrictive temperature (Fig.
5B; L.C.R. and S.L.J., unpublished). Plasma membrane fluorescence
of GFP-yck2
528-540p is dramatically reduced in favor of both soluble
and particulate cytosolic fluorescence
(Fig. 7).
The Yck1p C-terminus directs the Yck2p catalytic domain to the plasma
membrane via the secretory pathway
Although Yck1p and Yck2p are functionally redundant, it has not been
demonstrated directly that Yck1p is plasma membrane localized. Further,
although Yck2p and Yck1p share high sequence similarity in the catalytic
domain, and the C-terminal sequences of both proteins are Gln-rich, the
C-terminal domains are divergent in sequence
(Fig. 9A). Only the final 12
amino acids are markedly similar in sequence (83% identity). These include the
two terminal Cys residues, and six of the residues defined by the
yck2528-540 allele as required for both modification and
targeting. The additional sequences in the Yck2p C-terminus that are important
for Yck2p membrane association and plasma membrane targeting are not present
in Yck1p, and the Gln-rich sequences of Yck2p do not appear to influence
targeting greatly. These observations raise two questions, whether Yck1p is
modified in the same AKR1-dependent manner as Yck2p, and whether
Yck1p is targeted to the plasma membrane via the same route as Yck2p.
|
Because a GFP fusion to the N-terminus of Yck1p was neither functional nor readily detectable (L.C.R., unpublished), we generated a chimera between Yck1p and Yck2p (see Materials and Methods; Fig. 3A). GFP and the first 396 residues of Yck2p, including the entire catalytic domain, were fused to the C-terminal domain of Yck1p (residues 389 to 538). This chimera was functional as a Yck protein, as demonstrated by complementation of the yckts mutant when expressed from the YCK2 promoter (Fig. 5B). Consistent with this result, fluorescence of the chimera was detected at the plasma membrane (Fig. 9B, left panel), as for the wild-type GFP-Yck2p fusion.
We tested whether the GFP-Yck2/Yck1p chimera requires Akr1p for membrane
association by introducing a low copy plasmid containing the chimeric gene
into an akr1/akr1
strain. As shown in
Fig. 9B (right panel),
fluorescence of the chimera in this strain is not associated with the plasma
membrane (or any membrane), as for the wild-type GFP-Yck2 fusion protein in
the akr1 strain, or for the GFP-Yck2-Cys545,546Ser fusion
protein in any strain. Thus, the Yck1 protein also appears dependent on Akr1p
for modification that allows membrane association.
We also tested whether the divergent Yck1p C-terminus is sufficient for plasma membrane targeting via the secretory pathway. Plasmid pL240, containing the chimera behind the GAL1 promoter, was introduced into sec23 and sec9 strains. Induction of expression at permissive and non-permissive temperatures was carried out as for the GFP-Yck2 protein. As expected, the GFP-Yck2/Yck1p chimera is plasma membrane-associated in the sec9 mutant at its permissive temperature (Fig. 9C), as it is in the sec23 mutant at permissive temperature (L.C.R., unpublished). However, in neither sec mutant did the chimera reach the plasma membrane at restrictive temperature (Fig. 9C). Thus, targeting of this chimera is identical to that of intact GFP-Yck2p, even though there is no obvious sequence similarity in regions of the C-terminus required for Yck2p plasma membrane targeting.
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Discussion |
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The Yck2p C-terminal Cys residues are essential for membrane association.
Our results with the farnesylated Yck2-CIISp variant demonstrate that
hydrophobic modification at the C-terminus is sufficient to allow membrane
association. It was proposed that the two terminal Cys residues on Yck2p are
modified by geranylgeranylation, based on the conditions needed to extract
Yck2p from membranes and on the fact that the only other proteins that carry
two terminal Cys residues, Rab proteins, are modified on both Cys residues by
the GGTase type II. However, there is no direct evidence for this, and there
is now strong evidence that Yck2p is modified by palmitoylation. The Yck2
protein is neither labeled with palmitate nor membrane associated in cells
lacking the Akr1 protein (Feng and Davis,
2000; Roth et al.,
2002
). We have demonstrated here that the Yck1p C-terminal domain
also confers dependence on Akr1 for membrane association. The Akr1 protein
shares sequence similarity with the Erf2 protein, which is required for
palmitoylation of Ras2p (Bartels et al.,
1999
), and Yck2p can be palmitoylated in vitro in an
Akr1p-dependent fashion (Roth et al.,
2002
). These results indicate that one or both of the Cys residues
of both Yck1p and Yck2p are palmitoylated.
Yck1p and Yck2p provide additional examples of palmitoylated proteins
targeted to the inner surface of the plasma membrane. However, unlike some
palmitoylated proteins (Resh,
1999), it is not likely that Yck2p modification occurs at the
plasma membrane. Our results show that secretory membrane association of Yck2p
occurs soon after synthesis. The presence of the terminal Cys residues is
required for this association, suggesting that palmitoylation is a
prerequisite for association. The possibility that the Cys residues are
required in and of themselves for membrane association, for example, as
recognition determinants for a membrane targeting factor, is not ruled out by
our data. However, the observations that GFP-Yck2-CIISp is targeted normally
and retains full biological function argue against this possibility.
The apparently normal targeting of the Yck2-CIISp variant also addresses
another issue. Palmitoylation has been demonstrated to influence
protein-protein interactions (Dunphy and
Linder, 1998; Resh,
1999
), and so it seemed possible that substitution of a
farnesylation signal on Yck2p could impair secretory pathway targeting. This
does not appear to be the case, arguing that modification serves to promote
membrane association rather than targeting for Yck2p. Finally, the Yck2-CIISp
results, along with the results of our deletion analysis, indicate that
sequences in the C-terminus other than the Cys residues are important for
plasma membrane targeting. The Yck2p sequences that are required for
modification and targeting lie within the final 47 residues of the protein,
with the final 28 residues most important for modification. There is no known
consensus sequence for palmitoylation, and database searches with the Yck2p
terminal 28 residue sequence yielded no significant matches.
Two C-terminal deletion mutants had the strongest effect on Yck2p membrane
association. The GFP-yck2519-527 protein is not visible at any
membranes, but this variant is present at very low abundance, and lack of
modification may reflect a very short half-life of the protein. We have no
explanation for the low abundance of this mutant protein.
GFP-yck2
519-527 does not contain any known degradation signal, and the
possibility that lack of modification affects stability is ruled out by the
observations that the Cys545,546Ser mutant protein, and the
wild-type protein in the akr1 mutant, accumulate to wild-type levels.
The GFP-yck2
528-540 protein, by contrast, demonstrates marked
impairment of membrane association without effect on protein levels. Very
little of this protein is detectable on internal membranes and biological
function is lacking. It is likely that residues recognized by the palmitoyl
transferase lie within this deleted region.
The yck2499-518 deletion shows very little effect on
biological function or protein levels, but the mutant protein is less
efficiently transported to the plasma membrane. There is also an increased
pool of apparently soluble protein for this variant. Therefore, this protein
could be impaired for modification and/or targeting.
Yck1p and Yck2p are functionally redundant
(Robinson et al., 1992;
Wang et al., 1992
) and a
chimera with Yck2p catalytic domain and Yck1p C-terminal domain is functional
and is targeted to the plasma membrane as is intact Yck2p. However, outside
the catalytic domain, the two proteins share little in common aside from
C-terminal Gln-rich sequences. Beyond the last protein kinase subdomain, only
the first eight and the final 12 residues share identity. Shared residues
closest to the Cys residues, deleted in yck2
528-540p, are required for
membrane association, probably for modification. The residues deleted in
yck2
499-518p are unique to Yck2p and are important for plasma membrane
targeting. These residues are similar neither to sequences in the Yck1p tail
nor to any other protein sequence from yeast or higher eukaryotes. Therefore,
we predict that some sequence in the Yck1p C-terminus is functionally similar
to this Yck2p sequence. Structure rather than primary sequence could be most
important for recognition and targeting, but the C-terminus overall, and this
sequence in particular, has little predicted secondary structure.
Identification of targeting factor(s) and definition of a sequence in Yck1p
that functions similarly to the Yck2p sequence deleted in yck2
499-518p
will be the first steps in determining how such factor(s) can recognize two
very different sequences.
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Acknowledgments |
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References |
---|
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---|
Apolloni, A., Prior, I. A., Lindsay, M., Parton, R. G. and
Hancock, J. F. (2000). H-ras but not K-ras traffics to the
plasma membrane through the exocytic pathway. Mol. Cell
Biol. 20,2475
-2487.
Bankaitis, V. A., Malehorn, D. E., Emr, S. D. and Greene, R. (1989). The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108,1271 -1281.[Abstract]
Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M. and Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77,895 -907.[Medline]
Bartels, D. J., Mitchell, D. A., Dong, X. and Deschenes, R.
J. (1999). Erf2, a novel gene product that affects the
localization and palmitoylation of Ras2 in Saccharomyces cerevisiae.
Mol. Cell Biol. 19,6775
-6787.
Botstein, D., Amberg, D., Mulholland, J., Huffaker, T., Adams, A., Drubin, D. and Stearns, T. (1997). The yeast cytoskeleton. In The Molecular and Cellular Biology of the Yeast Saccharomyces, Cell Cycle and Cell Biology, Vol.3 (ed. J. R. Pringle, J. R. Broach and E. W. Jones), pp. 1-90. Plainview, NY: Cold Spring Harbor Laboratory Press.
Brennwald, P., Kearns, B., Champion, K., Keranen, S., Bankaitis, V. and Novick, P. (1994). Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell 79,245 -258.[Medline]
Brockman, J. L. and Anderson, R. A. (1991).
Casein kinase I is regulated by phosphatidylinositol 4,5-bisphosphate in
native membranes. J. Biol. Chem.
266,2508
-2512.
Carmel, G., Leichus, B., Cheng, X., Patterson, S. D., Mirza, U.,
Chait, B. T. and Kuret, J. (1994). Expression, purification,
crystallization and preliminary X-ray analysis of casein kinase-1 from
Schizosaccharomyces pombe. J. Biol. Chem.
269,7304
-7309.
Choy, E., Chiu, V. K., Silletti, J., Feoktistov, M., Morimoto, T., Michaelson, D., Ivanov, I. E. and Philips, M. R. (1999). Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98,69 -80.[Medline]
Davis, N. G., Horecka, J. L. and Sprague, J. G. F., III (1993). Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors. J. Cell Biol. 122,53 -65.[Abstract]
DeMaggio, A. J., Lindberg, R. A., Hunter, T. and Hoekstra, M. F. (1992). The budding yeast HRR25 gene product is a casein kinase I isoform. Proc. Natl. Acad. Sci. USA 89,7008 -7012.[Abstract]
Desnoyers, L., Anant, J. S. and Seabra, M. C. (1996). Geranylgeranylation of Rab proteins. Biochem. Soc. Trans. 24,699 -703.[Medline]
Dunphy, J. T. and Linder, M. E. (1998). Signalling functions of protein palmitoylation. Biochim. Biophys. Acta. 1436,245 -261.[Medline]
Eakle, K. A., Bernstein, M. and Emr, S. D. (1988). Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product. Mol. Cell. Biol. 8,4098 -4109.[Medline]
Egerton, M., Zueco, J. and Boyd, A. (1993). Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: subcellular distribution of a protein required for yeast protein secretion. Yeast 9,703 -713.[Medline]
Feng, Y. and Davis, N. G. (2000). Akr1p and the
type I casein kinases act prior to the ubiquitination step of yeast
endocytosis: Akr1p is required for kinase localization to the plasma membrane.
Mol. Cell. Biol. 20,5350
-5359.
Gietz, D., St Jean, A., Woods, R. A. and Schiestl, R. H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425.[Medline]
Givan, S. A. and Sprague, G. F., Jr (1997). The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors. Mol. Biol. Cell 8,1317 -1327.[Abstract]
Gonzalo, S. and Linder, M. E. (1998). SNAP-25
palmitoylation and plasma membrane targeting require a functional secretory
pathway. Mol. Biol. Cell
9, 585-597.
Gross, S. D. and Anderson, R. A. (1998). Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family. Cell Signal 10,699 -711.[CrossRef][Medline]
Hicke, L., Zanolari, B. and Riezman, H. (1998).
Cytoplasmic tail phosphorylation of the -factor receptor is required
for its ubiquitination and internalization. J. Cell
Biol. 141,349
-358.
Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. J. and Heffron, F. (1991). HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA. Science 253,1031 -1034.[Medline]
Kao, L.-R., Peterson, J., Ji, R., Bender, L. and Bender, A. (1996). Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16,168 -178.[Abstract]
Lewis, M. J., Nichols, B. J., Prescianotto-Baschong, C.,
Riezman, H. and Pelham, H. R. B. (2000). Specific retrieval
of the exocytic SNARE Snc1p from early endosomes. Mol. Biol.
Cell 11,23
-38.
Lobo, S., Greentree, W. K., Linder, M. E. and Deschenes, R. J. (2002). Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J. Biol. Chem.
Loranger, S. S. and Linder, M. E. (2002).
SNAP-25 traffics to the plasma membrane by a syntaxin-independent mechanism.
J. Biol. Chem. 277,34303
-34309.
Marchal, C., Haguenauer-Tsapis, R. and Urban-Grimal, D.
(2000). Casein kinase I-dependent phosphorylation within a PEST
sequence and ubiquitination at nearby lysines signal endocytosis of yeast
uracil permease. J. Biol. Chem.
275,23608
-23614.
Novick, P., Field, C. and Schekman, R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21,205 -215.[Medline]
Panek, H. R., Stepp, J. D., Engle, H. M., Marks, K. M., Tan, P.
K., Lemmon, S. K. and Robinson, L. C. (1997). Suppressors of
YCK-encoded yeast casein kinase 1 deficiency define the four subunits
of a novel clathrin AP-like complex. EMBO J.
16,4194
-4204.
Poon, P. P., Cassel, D., Spang, A., Rotman, M., Pick, E.,
Singer, R. A. and Johnston, G. C. (1999). Retrograde
transport from the yeast Golgi is mediated by two ARF GAP proteins with
overlapping function. EMBO J.
18,555
-564.
Pryciak, P. M. and Hartwell, L. H. (1996). AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction. Mol. Cell. Biol. 16,2614 -2626.[Abstract]
Raths, S., Rohrer, J., Crausaz, F. and Riezman, H. (1993). end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae.J. Cell Biol. 120,55 -65.[Abstract]
Resh, M. D. (1999). Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451, 1-16.[Medline]
Roach, P. J. (1990). Control of glycogen synthase by hierarchal protein phosphorylation. FASEB J. 4,2961 -2968.[Abstract]
Robinson, L. C., Hubbard, E. J. A., Graves, P., DePaoli-Roach, A. A., Roach, P. J., Kung, C., Haas, D. W., Hagedorn, C. H., Goebl, M., Culbertson, M. R. et al. (1992). Yeast casein kinase I homologues: an essential gene pair. Proc. Natl. Acad. Sci. USA 89,28 -32.[Abstract]
Robinson, L. C., Menold, M. M., Garrett, S. and Culbertson, M. R. (1993). Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis. Mol. Cell. Biol. 13,2870 -2881.[Abstract]
Robinson, L. C., Bradley, C., Bryan, J. D., Jerome, A., Kweon,
Y. and Panek, H. R. (1999). The Yck2 yeast casein kinase 1
isoform shows cell cycle-specific localization to sites of polarized growth
and is required for proper septin organization. Mol. Biol.
Cell 10,1077
-1092.
Roth, A. F., Feng, Y., Chen, L. and Davis, N. G.
(2002). The yeast DHHC cysteine-rich domain protein Akr1p is a
palmitoyl transferase. J. Cell Biol.
159, 23-28.
Sherman, F., Fink, G. R. and Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Taylor, R. G., Walker, D. C. and McInnes, R. R. (1993). E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res. 21,1677 -1678.[Medline]
TerBush, D. R. and Novick, P. (1995). Sec6, Sec8 and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130,299 -312.[Abstract]
Tuazon, P. T. and Traugh, J. A. (1991). Casein kinase I and II- multipotential serine kinases: structure, function and regulation. Adv. Second Messenger Phosphoprotein Res. 23,123 -164.[Medline]
Vancura, A., O'Connor, A., Patterson, S. D., Mirza, U., Chait, B. T. and Kuret, J. (1993). Isolation and properties of YCK2, a Saccharomyces cerevisiae homolog of casein kinase I. Arch. Biochem. Biophys. 305, 47-53.[CrossRef][Medline]
Vancura, A., Sessler, A., Leichus, B. and Kuret, J.
(1994). A prenylation motif is required for plasma membrane
localization and biochemical function of casein kinase I in budding yeast.
J. Biol. Chem. 269,19271
-19278.
Wang, P.-C., Vancura, A., Mitcheson, T. G. and Kuret, J. (1992). Two genes in Saccharomyces cerevisiae encode a membrane bound form of casein kinase-1. Mol. Biol. Cell 3,275 -286.[Abstract]
Wang, X., Hoekstra, M. F., DeMaggio, A. J., Dhillon, N., Vancura, A., Kuret, J., Johnston, G. C. and Singer, R. A. (1996). Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase. Mol. Cell. Biol. 16,5375 -5385.[Abstract]
Xu, R.-M., Carmel, G., Sweet, R. M., Kuret, J. and Cheng, X. (1995). Crystal structure of casein kinase-1, a phosphate directed protein kinase. EMBO J. 14,1015 -1023.[Abstract]