(Received for publication, June 26, 1995; and in revised form, October 6, 1995)
From the
The core of the vacuolar targeting signal of yeast
carboxypeptidase Y (CPY) is recognized by the receptor Vps10p and
consists of four contiguous amino acid residues,
Gln-Arg-Pro-Leu
, near the amino terminus of
the propeptide (Valls, L. A., Winther, J. R., and Stevens, T. H.(1990) J. Cell Biol. 111, 361-368; Marcusson, E. G.,
Horazdovsky, B. F., Cereghino, J. L, Gharakhanian, E., and Emr, S.
D.(1994) Cell 77, 579-586). In order to determine the
sequence specificity of the interaction with the sorting receptor,
substitutions were introduced into this part of the propeptide by
semirandom site-directed mutagenesis. The efficiency of vacuolar
sorting by the mutants was determined by immunoprecipitation of CPY
from pulse-labeled cells. It was found that amino acid residues
Gln
and Leu
were the most important ones.
While it appears that Gln
is essential for proper
function, Leu
can be exchanged with the other hydrophobic
amino acid residues, isoleucine, valine, and phenylalanine. Tolerance
toward various substitutions for Arg
is fairly high, while
substitution of Pro
for uncharged amino acid residues also
resulted in only weak missorting. In addition to the low requirement
for sequence conservation, the position of the targeting element
relative to the amino terminus of the propeptide was analyzed and found
not to be critical.
Eukaryotic cells consist of a number of compartments, each with a unique set of proteins, allowing the cell to regulate spatially its catalytic activities. This requires an efficient system for directing each newly synthesized protein to its correct functional location within the cell.
The targeting signals are ultimately encoded in the
protein sequence, and these signals take many forms. Signals for
protein import into mitochondria, for example, lack any obvious
sequence homology but tend to form amphipatic -helices or
-sheets, which allow them to bind to the mitochondrial membrane,
whereafter specific recognition occurs (Hartl and Neupert, 1990, Baker
and Schatz, 1991). Similarly, NH
-terminal leader peptides,
which direct proteins into the ER (
)of eukaryotic cells and
function in translocation through the plasma membrane in prokaryotes,
also lack highly specific primary sequence determinants. Here more
general physical properties such as hydrophobicity and charge are
important for function (von Heijne, 1985). Conversely, targeting of
some proteins to peroxisomes occurs via a fairly well defined signal
consisting of three amino acid residues at the COOH terminus. The
tripeptide SKL at the COOH terminus of firefly luciferase was shown to
direct this protein into peroxisomes (Gould et al., 1987).
Various substitutions in this tripeptide signal have been made, some of
which are functional in peroxisomal import (Subramani, 1992).
The default route for soluble proteins in the secretory pathway is to the cell surface, i.e. they will be secreted if they lack any specific signals. Soluble ER proteins in Saccharomyces cerevisiae contain the COOH-terminal tetrapeptide HDEL, which allows them to be retained in the ER (Pelham, 1989). This is an example of a very specific signal, and recycling of HDEL-tagged proteins to the ER in yeast is mediated by a receptor, Erd2p (Lewis et al., 1990).
In mammalian cells, lysosomal targeting is mediated by the mannose
6-phosphate receptor and, ultimately, by the recognition of the
structural elements of the lysosomal proteins by the phosphotransferase
(Baranski et al., 1990). Although the yeast vacuole is
equivalent to the lysosome of higher eukaryotes in many respects, no
mannose 6-phosphate-mediated sorting mechanism has been found in yeast.
The soluble vacuolar hydrolases proteinase A and carboxypeptidase Y
(CPY) of S. cerevisiae contain vacuolar targeting information
in their NH-terminal propeptides (Johnson et al.,
1987, Valls et al., 1987, Klionsky et al., 1988). The
sorting of proteinase A and CPY was suggested to be receptor-mediated
since overexpression resulted in mislocalization (Rothman et
al., 1986, Stevens et al., 1986). Indeed, the sorting
receptor for CPY was recently identified as the VPS10 gene
product (Marcusson et al., 1994). The information necessary
and sufficient for vacuolar localization of CPY is encoded by a
sequence containing the tetrapeptide
Gln
-Arg-Pro-Leu
near the NH
terminus of the precursor (Valls et al., 1990). The
information for targeting of proteins to the vacuole of plant cells may
also be found in short peptide domains (Chrispeels and Raikhel, 1992),
but QRPL is the best characterized vacuolar targeting signal so far and
is therefore often used as a basis for defining and comparing with
other potential vacuolar targeting signals. None of the other known
vacuolar proteins in yeast contain a QRPL sequence. However, since only
mutations in the QRPL signal abolishing its function have been defined,
it is difficult to deduce a consensus sequence for comparison with
other systems.
Using degenerate oligonucleotides we have performed
extensive mutagenesis on the part of the PRC1 gene encoding
the QRPL signal and determined the efficiencies of sorting by
immunoprecipitation of newly synthesized CPY in pulse-chase
experiments. We found that many mutant forms of the QRPL signal are
indeed able to sort pro-CPY correctly to the vacuole. Furthermore, we
analyzed the importance of the distance of the QRPL signal from the
NH terminus of the propeptide and found that insertion of
up to four amino acid residues did not affect the sorting efficiency
seriously. These data together suggest a surprisingly low level of
requirement of sequence conservation for function of the recognition of
the sorting receptor.
Other libraries were created by insertion of oligonucleotide
mixtures containing four extra codons inserted between positions 22 and
23. In order to eliminate stop codons but still obtain a fairly random
collection of mutants, each of the additional new codons started with
an adenine followed by two random nucleotides. This oligonucleotide
mixture was ligated, and total plasmid DNA was isolated from a pool of
150 individual E. coli transformants. Plasmid DNA was
reintroduced into E. coli, and plasmid DNA from five of the
transformants was used for sequencing. Oligonucleotides with several
desired mutations or insertions were synthesized individually and
cloned as described above. Mutations were verified by DNA sequencing.
Plasmid pJW1588 contains an allele of PRC1, PRC1-2112, with the mutations T15A and N368Q on a vector similar to pLV9 (Winther et al., 1991). These mutations remove the first and the last glycosylation site, and the resulting mutant protein is called aBCd-CPY. A SalI-PvuII fragment containing this mutant allele was subcloned into pRS315 (a LEU2 CEN vector; Sikorski and Hieter(1989)), resulting in pFV410.
The plasmid pFV127, containing the fusion between PRC1 and SUC2, was constructed as follows: a BamHI-SmaI fragment containing the first 156 codons of the PRC1 gene was subcloned in pSEYC306 (Johnson et al., 1987), resulting in pFV50. A SalI fragment of pSEYC306 was subcloned in pRS315 opened with SalI and XhoI, so that the 5` end of the SUC2 gene was nearest to the remaining SalI site, resulting in pFV99. Subsequently, a SmaI-SalI fragment from pFV50 was subcloned in pFV99 opened with SmaI and SalI.
Figure 1: Sequence containing the QRPL signal. A XhoI restriction site necessary for the cassette mutagenesis was introduced. The site of the signal peptidase cleavage site is indicated by sp.
As an initial approach we wished to isolate mutants in the QRPL
signal that were proficient in sorting. Thus, libraries of mutant
plasmids were constructed at each of the positions Leu
through Leu
(pools Xaa
through
Xaa
) and more than 500 individual E. coli transformants were obtained from each oligonucleotide mixture.
Cells from all transformant colonies of each ligation were pooled, and
plasmid DNA was isolated from the pools and used for transformation of
a
prc1 yeast strain. Mutants were characterized according
to two criteria: 1) the presence of intracellular CPY activity,
determined by a plate overlay assay, and 2) secretion of CPY, detected
by colony immunoblots. Mutants strongly deficient in sorting, as well
as non-sense mutants, were not expected to give intracellular activity,
while missorting mutants stain positive in the immunoblot. From each
collection, 100 transformants were replica-plated and tested according
to both criteria. This gave an initial indication as to the specificity
of the sorting signal at each position, as well as the frequency of
nonsense and frameshift mutations that would lead to absence of both
activity and antigen. The number of colonies without CPY production
varied between 5 and 7% in all collections, which corresponds to the
expected frequency of stop codons. The most important result of this
initial screen was that only two of the 100 yeast Xaa
transformants showed a wild-type phenotype. Screening of the
Xaa
, Xaa
, and Xaa
pools showed a
higher frequency (5-15%) of plasmids that gave rise to a
wild-type or quasi-wild-type phenotype, while all plasmids from the
Xaa
pool appeared to give a wild-type phenotype.
Sequencing of the plasmid DNA from the two positive clones of the
Xaa
pool showed that the wild-type phenotype was in both
due to a Gln codon.
All plasmids giving what appeared to be a
wild-type phenotype in the plate assays were sequenced, and to get a
broader view of the specificity, several randomly chosen plasmids were
sequenced from each library. The sorting efficiency of each mutant was
determined quantitatively by S pulse-chase labeling and
immunoprecipitation. Thus, exponentially growing cultures were labeled
with [
S]methionine/cysteine, and CPY antigens
were immunoprecipitated from intracellular and extracellular fractions.
Samples were subsequently subjected to SDS-polyacrylamide gel
electrophoresis, and autoradiograms were prepared using a
PhosphorImager. Fig. 2shows representative immunoprecipitations
of mutants with different phenotypes. CPY that is correctly localized
to the vacuole is processed to the mature form of 61 kDa. However,
mislocalized pro-CPY is not matured and is found extracellularly as the
precursor form of 69 kDa. Wild-type pro-CPY is sorted to the vacuole
and converted to its mature form. However, a small fraction, around 5%,
is mislocalized and is seen extracellularly as the pro-CPY precursor
form (Fig. 2, lane 2). This level of missorting of the
wild type is normal (Stevens et al., 1986; Valls et
al., 1987, 1990). Appearance of CPY antigen extracellularly is not
due to cell lysis, since no mature CPY is found extracellularly.
Radioactivity in the CPY-specific bands was detected and quantified
using storage phosphor technology (Johnston et al., 1990). The
ratios of intra- and extracellular CPY were determined by
quantification of at least two independent immunoprecipitations. The
amounts of intracellular CPY relative to the total amounts synthesized
are depicted in Fig. 3together with data from an earlier study
(Valls et al., 1990). Our determinations of the missorting
phenotype of mutations Q24S and R25G are in good agreement with those
obtained previously by Valls et al.(1990). The differences, 7%
and 5% for Q24S and R25G, respectively, reflect the internal variation
found in our own measurements. In addition to those clones selected for
analysis on the basis of wild-type sorting in the plate screen and
randomly selected clones, we constructed some directed mutants that
were judged to supplement the collection well.
Figure 2:
Immunoprecipitation of CPY antigen from
selected mutants. Cells expressing three mutant PRC1 alleles
and the wild type (WT) were labeled for 20 min with S-labeled amino acids and chased with nonlabeled amino
acids and sulfate for 60 min. Intracellular (I) and
extracellular (E) material were separated, and CPY antigen was
immunoprecipitated. The labeled precipitates were subsequently
subjected to 8% SDS-polyacrylamide gel electrophoresis. Within the
duration of the chase period all CPY reaches its final
destination.
Figure 3:
Sorting efficiency of mutant forms of
pro-CPY. Bars indicate the percentage of labeled CPY antigen
found intracellularly relative to the total amount of labeled CPY,
intracellularly and extracellularly, after a 20-min S
pulse followed by a 60-min chase. Shaded bars indicate mutants
that have been characterized in this study, while those indicated by hatched bars are from Valls et al.(1990). wt, wild type.
All the mutations in
Gln lead to more than 50% missorting, even the
conservative Q24N mutation, which was introduced by directed
mutagenesis. The combined results from the immunoprecipitation and the
screening of colonies on plates strongly suggest that only glutamine
will function well in sorting at this position.
In general,
mutations in Arg do not appear to have a strong effect on
sorting. Most severe is the R25D mutation, which results in secretion
of 48% of the newly synthesized pro-CPY. All the other mutations have a
sorting efficiency between 74 and 95%. This is in good agreement with
the large number of mutations (Asn, Asp, Glu, Gly, Lys, and Val) scored
as wild-type or quasi-wild-type in the initial plate screen.
Mutations in Pro give rise to a wide spectrum of
phenotypes. P26R leads to missorting of almost all of the newly
synthesized pro-CPY, whereas the P26S mutation still localizes 73% of
the pro-CPY to the vacuole. Mutants with hydrophobic amino acid
residues (Phe, Val, Leu, Ala) at this position show better than 80%
sorting. Only proline (3 clones) and serine (one clone) were selected
as correctly sorting in the plate assays of the random mutants at this
position.
All mutations in Leu except those with bulky
hydrophobic residues give very severe missorting phenotypes. More than
95% of the L27A mutant pro-CPY is secreted, while the hydrophobic
residues (Phe, Ile, Val) give rise to mutant forms that are sorted with
wild-type efficiency.
Since the initial screen had shown that mutations at position 23 had very little effect on sorting, only three randomly selected mutants were analyzed quantitatively. As expected, the missorting phenotypes of these were not strong (Fig. 3).
Replacing the codon for Gly by random DNA sequence was
not possible using random substitutions since this codon forms part of
the XbaI site used for ligating the mutagenic oligonucleotides (Fig. 1). Consequently, directed mutations were introduced to
exchange Gly
with arginine, phenylalanine, or aspartic
acid residues. Pulse-chase experiments showed that even these radical
changes had limited effect, the strongest being Asp
, which
gives missorting of 21% of the newly synthesized pro-CPY (Fig. 3).
In a more radical approach, four extra codons were introduced between codons 22 and 23. Each extra codon started with adenine followed by two random nucleotides to avoid stop codons. Five plasmids resulting from this mutagenesis were sequenced (Table 1), and the sorting efficiencies of the respective CPY mutant proteins were determined in pulse-chase experiments. One of the mutants secretes 25% of the total amount synthesized, which is significantly more than the wild type. The secretion of the other of four mutant CPY proteins, however, is only slightly higher than that of the wild type.
In parallel to this approach we tested mutations P26D, L27A, L27G, and L27R, which exhibited the strongest missorting phenotypes, using an approach very similar to that described by Valls et al. (1990), analyzing the competition between aberrant glycoforms of CPY and the QRPL mutant forms in immunoprecipitation experiments (not shown). This approach also suggested that receptor poisoning is a phenomenon that cannot be brought about by mutation of a single residue in the QRPL signal. Thus, we find it unlikely that single amino acid substitutions can lead to poisoning of the receptor.
The ligand-receptor pair, which is central to the present
discussion, represents the first and best characterized member of a new
class of intracellular sorting mechanisms. It has long been known that
the signal for pro-CPY sorting to the vacuole resides in the proregion
and that a sequence containing Gln-Arg-Pro-Leu
is both necessary and sufficient for this function (Valls et
al., 1987, 1990, Johnson et al., 1987). Through the use
of genetic screens, this observation eventually led to the
identification of the VPS10 gene, which encodes the receptor
directly involved in QRPL recognition. The VPS10 gene product
(Vps10p) is a very large type I transmembrane protein (1577 amino acid
residues) with a short COOH-terminal cytoplasmic tail (164 amino acid
residues). In cell fractionation as well as functional studies, it
localizes to the distal Golgi apparatus together with the Kex2 protease
(Marcusson et al., 1994, Graham et al., 1991).
Chemical cross-linking experiments have shown that Vps10p interacts
specifically with the QRPL signal, both in a pro-CPY context and in an
invertase fusion context (Marcusson et al., 1994). Mutations
that resulted in missorting of pro-CPY had been found in each of the
QRPL residues (Valls et al., 1990), and it was directly shown
that the Q24K mutant protein did not chemically cross-link to Vps10p
(Marcusson et al., 1994). However, the level of sequence
conservation required for productive pro-CPY-Vps10p interaction has not
been investigated. Since the QRPL system has been described in such
detail it has to some extent been regarded as a paradigm for comparison
to other non-carbohydrate-dependent lysosomal/vacuolar sorting systems.
It has been suggested that the QRPL signal might be a part of a larger
``consensus sequence'' for lysosomal sorting both in mammals
and yeast (McIntyre et al., 1994). The original screen would
not address the validity of such a consensus since the mutants were
identified by their inability to confer vacuolar sorting to pro-CPY, i.e. no functional mutants were isolated (Valls et
al., 1990). This has also hampered the identification of other
putative QRPL sequences by homology searches, as the significance of
individual residues was not known. In the present work we have
attempted to approach a consensus sequence for the specificity of the
receptor-ligand interaction.
One of the results of the present study
is the identification of the pivotal role of Gln in the
sorting process. All mutations at this position showed severe
missorting phenotypes, and we failed in our attempts to isolate, by
activity stain and immunoblotting, mutants that were able to
functionally substitute for Gln
. We therefore conclude
that this amino acid residue is essential for the proper recognition of
the targeting element. There seems to be a similar importance for the
structural conservation of Leu
, although several
hydrophobic residues (Phe, Val, and Ile) were functional. Because of
the mutational approach taken, we cannot exclude the possibility that
tryptophan or methionine residues, encoded by rare codons, might also
function.
Although missorting mutations can be found at positions
Arg, Pro
, and Gly
, the nature of
the residues at these positions is clearly of lesser importance. The
discussion of the phenotypes of these mutants to some extent depends on
the definition of missorting. One should realize that pro-CPY sorting
is never 100% efficient; we typically find around 5% of the newly
synthesized wild-type pro-CPY to be missorted. This might be due to
problems of stoichiometry at the site of interaction, but it might also
be due to an inherent lack of affinity. It is conceivable that CPY
might also, under some conditions, be beneficial for extracellular
peptide hydrolysis. Thus, there may not have been strong evolutionary
pressure for 100% sorting efficiency.
In any event, the
substitutions for Arg in several cases lead to a clearly
wild-type phenotype. However, it is not easy to rationalize the
phenotypes from structural considerations since not only the
conservative change to lysine but also radical changes to the
hydrophobic amino acids leucine and valine and the polar glutamine fail
to affect sorting. In addition, most other mutations obtained have only
fairly weak missorting phenotypes, less than 30% being secreted in most
strains. Only a substitution by aspartate appears to affect sorting
strongly. Most of the mutants characterized at this position were
selected on the basis of their proficiency in sorting. Thus, the
relatively small number of strong missorting mutants reflects the
selection procedure employed.
Mutations leading to amino acid
substitutions for Leu had a similar effect (Fig. 3). On immunoblots, no mutants could be identified that
showed severe missorting. Thus, randomly chosen mutations were tested
in immunoprecipitations, and only weak phenotypes were found. These
findings support the notion that Leu
is not very
important.
Proline is important for specific tertiary structures in
many protein contexts. It is therefore surprising that many of the
randomly selected mutations at position 26 only had limited effect.
There appears to be a correlation between charge and missorting
phenotype and a clear preference for hydrophobic residues. The
phenotype of the Asp mutant (21% missorting) could suggest
that Gly
is just as much a part of the sorting signal as
Phe
. Considering that glycine also has unique structural
features, one might have expected more severe missorting phenotypes. As
these two examples show, there is a remarkable insensitivity toward the
tertiary structural environment of the QRPL signal. This notion is
supported by the observation that fairly short fusions (30 propeptide
residues) of the QRPL signal to invertase are able to direct this
secreted protein to the vacuole (Johnson et al., 1987).
Furthermore, the deletion of large fractions of the propeptide
downstream of Asp
had no effect on sorting (Ramos et
al., 1994). Indeed, the combined structural and biochemical
analysis of the propeptide suggests that it may have a highly flexible
and dynamic structure (Sørensen et al., 1993).
Considering the essential role of the propeptide in folding (Winther and Sørensen, 1991; Ramos et al., 1994), mutagenesis in the propeptide could result in aberrantly folded pro-CPY molecules, and these misfolded molecules might therefore not be recognized by the sorting receptor. It has been shown that mutants having amino acids 27-31 deleted are folded correctly and exit from the ER with normal efficiency (Valls et al. 1987). This suggested that changes in this part of the propeptide would not seriously affect folding. We examined one representative mutant for each position with shorter pulse and chase times and observed maturation half-lives shorter than 15 min (data not shown). The insertion mutants containing four extra residues, however, did show a minor folding defect suggested by a half-time of ER exit of about 30 min. This somewhat extended half-time of maturation also resulted in a small amount, less than 10%, of pro-CPY in the intracellular fraction after a 1-h chase. In all the other experiments there was no detectable pro-CPY left in the intracellular fractions after a 1-h chase. After the 20-min labeling the relative amount of CPY in the proform is typically 20-40%. The minimal detection limit is 2.5%. This also shows that the half-time of maturation for all the single-amino acid mutants is less than 15 min. One should also bear in mind that it is unlikely that misfolded pro-CPY would escape the ER. Indeed, deletions in the propeptide that result in reduced efficiency of folding do not result in missorting (Ramos et al. 1994). Finally, the folded mature domain of CPY is not necessary for recognition by the receptor since CPY-invertase hybrid proteins containing as little as 30 amino acid residues of CPY are sorted efficiently (Johnson et al. 1987).
The QRPL
signal does not conform to a SXX+XL
consensus (where + is a positively charged residue and X is any amino acid residue) as suggested previously (McIntyre et al., 1994). The initial S is supposed to correspond to
Ser in pro-CPY, but the present study shows that this
residue is not important, while Gln
is. Apart from the
apparently essential function of Gln
, one should probably
be cautious with defining a consensus. Although there are clear
tendencies to suggest specificity for hydrophobic residues at position
27, it is difficult to rationalize the missorting phenotypes for the
other residues. It is surprising that the specificity is not more
stringent, especially since there does not appear to be any strict
requirement for conservation of distance to the NH
terminus. The specificity may to some extent be determined by the
exposure of the signal, possibly in a random coil. Such structures are
probably not common among proteins en route to the harsh
extracellular environment. Along these lines we suggest that the
somewhat reduced sorting efficiency seen in some of the insertion
mutants (Table 1) is more likely to be due to steric interference
than to disruption of a recognition element. The important and somewhat
surprising conclusion of the present work is that there does not appear
to be a conventional ``consensus sequence'' for ligands of
Vps10p. It will thus be difficult to identify ligands by sequence
alignment and, in a comparison across species, this problem would be
further enhanced by slight changes in specificity likely to occur even
between related ligand-receptor pairs.