(Received for publication, November 9, 1995; and in revised form, January 12, 1996)
From the
The sorting of the yeast proteases proteinase A and
carboxypeptidase Y to the vacuole is a saturable, receptor-mediated
process. Information sufficient for vacuolar sorting of the normally
secreted protein invertase has in fusion constructs previously been
found to reside in the propeptide of proteinase A. We found that
sorting of such a hybrid protein is dependent on the vacuolar
protein-sorting receptor Vps10p. This was unexpected, as strains
disrupted for VPS10 sort more than 85% of the proteinase A to
the vacuole. Consistent with a role for Vps10p in sorting of proteinase
A, we found that 1) overproduction of Vps10p suppressed the missorting
phenotype associated with overproduction of proteinase A, 2)
overproduction of proteinase A induced missorting of carboxypeptidase
Y, 3) vacuolar sorting of proteinase A in a vps10 strain
was readily saturated by modest overproduction of proteinase A, and 4)
Vps10p and proteinase A interact directly and specifically as shown by
chemical cross-linking. Interestingly, overexpression of two
telomere-linked VPS10 homologues, VTH1 and VTH2 suppressed the missorting phenotypes of a
vps10 strain. However, disruption of the VTH1 and VTH2 genes did not affect the sorting of proteinase A. We conclude that
proteinase A utilizes at least two mechanisms for sorting, a
Vps10p-dependent path and a Vth1p/Vth2p/Vps10p-independent path.
The yeast vacuole contains a number of soluble hydrolases that
are delivered to this organelle via the endoplasmic reticulum and the
Golgi complex. Thus, both vacuolar and secretory proteins transit
through these early compartments of the secretory pathway on their way
to their final destination. Sorting takes place in a late
subcompartment of the Golgi complex, the trans Golgi network,
where soluble vacuolar proteins are diverted to the late endosome by an
active, saturable
mechanism(1, 2, 3, 4) .
Carboxypeptidase Y (CPY) ()and proteinase A (PrA) have been
the model enzymes in most studies of the biosynthesis of soluble yeast
vacuolar proteins. These enzymes are synthesized as precursor forms
that upon arrival in the vacuole are activated by proteolytic removal
of an N-terminal propeptide. The vacuolar sorting signal of CPY is
located in the
propeptide(5, 6, 7, 8) , and the VPS10 gene encodes the receptor (Vps10p), which interacts with
this signal and is responsible for the sorting of CPY(9) .
Disruption of VPS10 results in complete mislocalization of CPY
but does not strongly affect vacuolar sorting of PrA, indicating that
PrA can be sorted to the vacuole by an alternate mechanism(9) .
The 54-amino acid propeptide of PrA can direct the normally periplasmic
enzyme invertase to the vacuole, indicating that it contains sorting
information(10) . More precise identification of this
propeptide-located sorting signal turned out to be difficult, as the
propeptide proved to be essential for folding, and thus also
endoplasmic reticulum exit, of PrA(11) . In a previous study,
the role of the PrA propeptide in folding of the enzyme was
investigated by random substitution of either the N-terminal or the
C-terminal half of the propeptide. In the subsequent screen for
functional mutant PrA forms, many were found to produce active PrA, and
these mutant PrA forms were also localized to the vacuole(12) .
This raised the possibility that the propeptide was not essential for the sorting of PrA, a hypothesis that we have investigated
further in the present study. We find that the PrA propeptide contains
a recognition site for Vps10p and that PrA can be sorted to the vacuole
by a Vps10p-dependent mechanism as well as a Vps10p-independent
mechanism. Furthermore, we describe a Vps10p homologue, Vth1p, that can
also function as sorting receptor for both PrA and CPY.
PrA activity was determined using an internally quenched fluorescent peptide substrate as described previously(11) .
Figure 1:
Overproduction of Vps10p suppresses the
PrA-missorting phenotype associated with overproduction of pro-PrA.
Cells were pulse labeled with S-labeled amino acids for 20
min and chased for 45 min. PrA antigen was immunoprecipitated from
intracellular (I) and extracellular (E) fractions.
Labeled protein was separated by 10% SDS-PAGE. The strain
(JHRY20-2C-
prc1) was devoid of CPY and expressed PEP4 from a centromere plasmid pBVH17 (WT +
CENPEP4
). CENPEP4
indicates
that this PEP4-carrying plasmid expresses 3-5-fold as
much PrA as the chromosomal copy of PEP4. The cells contained
a multicopy plasmid with (pVW197; lanes 3 and 4) or
without (pRS425; lanes 1 and 2) VPS10.
PseudoPrA is the product of the autoactivation of pro-PrA in the
extracellular media fraction (15) and is indicated by an asterisk.
Figure 2:
Sorting of pro-CPY is affected by
overproduction of pro-PrA. CPY was immunoprecipitated and prepared from
pulse labeled cells as described in the legend to Fig. 1using
8% SDS-PAGE. The intracellular material is indicated by I,
while extracellular material (medium + periplasm) is marked E. The cells expressed PEP4 at various gene dosages:
single chromosomal copy (WT), single copy + low copy centromere
plasmid pBVH17 (WT + CENPEP4), single copy
+ multicopy 2-µm plasmid pPA2 (WT + 2
µPEP4). The quantitation of CPY protein expressed as a
percentage of the total is shown below each lane.
Intensities of bands were quantitated using the PhosphorImager.
CENPEP4
indicates that this PEP4-carrying
plasmid expresses 3-5-fold as much PrA as the chromosomal copy of PEP4.
Figure 3:
A vps10 strain has a lower
capacity for PrA sorting than a wild-type strain. Cells were pulse
labeled with
S-labeled amino acids for 10 min and chased
for 30 min. PrA antigen was immunoprecipitated from intracellular (I) and extracellular (E) fractions. Labeled protein
was separated by 8% SDS-PAGE. Panel A, first and second lanes, a wild-type strain (SEY6210); third and fourth lanes, a wild-type strain (SEY6210) carrying a
centromere plasmid pSEYC306-PEP4 containing PEP4. Panel B, first and second lanes, a
vps10 strain (EMY3); third and fourth
lanes, a
vps10 strain (EMY3) carrying a centromere
plasmid pSEYC306-PEP4 containing PEP4.
Figure 5: Schematic representation of Vps10p and Vth1p. The 14 boxes in each molecule represent Asp boxes (23) with the sequence (S/T)XDXGX(T/S)(W/F). Domains 1 and 2 of each molecule are approximately 20% identical to each other. The spacing of the cysteines in the cysteine-rich motifs is perfectly conserved between the two proteins. These motifs are likely to be structural elements. The percentage of identity between the various domains of the proteins is indicated by the numbers between the molecules. Gray boxes indicate the transmembrane domains.
Figure 6:
Vth
proteins are expressed at very low levels. Cells were pulse labeled
with S-labeled amino acids for 30 min. Vps10p antigen was
immunoprecipitated from intracellular fractions. Labeled protein was
separated by 8% SDS-PAGE. Lane 1, wild-type strain (SEY6210); lane 2,
vps10 strain (EMY3); lane 3,
vps10 strain (EMY3) carrying a VTH1-containing
2-µm plasmid (pEMY106).
Figure 4:
Chemical cross-linking of Vps10p and
pro-PrA. Spheroplasts were pulse labeled with S-labeled
amino acids and lysed in a way that retained the integrity of
organelles. Lysates were then treated with the membrane-permeable
cross-linker dithio-bis(succinimidylpropionate), and treated
with Vps10p-specific antibodies. The precipitated immune complexes were
treated with a reducing and denaturing buffer to release cross-linked
proteins and antibodies. Finally, the samples were treated with
PrA-specific antibodies, and the precipitated proteins were analyzed by
SDS-PAGE. Lanes 1 and 2, strain SEY6210 (VPS10); lanes 3 and 4, strain EMY3 (
vps10); lanes 5 and 6, strain TVY1 (
pep4). The positions of the endoplasmic reticulum and
Golgi-modified forms of PrA are indicated by proPrA, and the
position of the mature enzyme is indicated by mPrA. For
molecular weight comparison, the rightmost lane shows all of
the PrA antigens.
Together, the data indicate that the 137 most N-terminal residues of pro-PrA contain at least two sorting signals, one in the C-terminal half of the propeptide (as was earlier proposed by Klionsky et al.(10) ) and one in the 59 most N-terminal residues of the mature enzyme region.
Because PrA is efficiently sorted to the vacuole in a strain disrupted for VPS10(9) , there must be a Vps10p-independent mechanism for PrA sorting. We constructed a fusion consisting of the entire pro-PrA sequence fused to invertase (PrA405-Inv). In order to allow correct folding of both enzymes, the two domains in the hybrid were separated by a linker peptide consisting of five glycine residues. Sorting of PrA405-Inv in strains lacking or containing Vps10p was investigated. In a VPS10 strain, 5% PrA405-Inv was secreted to the extracellular space. In contrast, in a strain lacking Vps10p, 60% of the invertase activity was secreted (Table 1). The presence of a considerable amount of PrA405-Inv inside the cells suggests that at least a portion of this fusion can be sorted to the vacuole via a VPS10-independent mechanism. The fact that strains expressing PrA405-Inv from a centromere plasmid produced as much PrA activity as a similar plasmid construct producing wild-type PrA (data not shown) suggests that the PrA domain of the hybrid protein folded efficiently into an active conformation and that the cell-associated material is targeted and activated normally in the vacuole.
Figure 7:
Vth1p can sort CPY and PrA to the vacuole.
Cells were pulse labeled with S-labeled amino acids for 10
min and chased for 30 min. CPY and PrA antigens were immunoprecipitated
from intracellular (I) and extracellular (E)
fractions. Labeled protein was separated by 8% SDS-PAGE. Panel
A, first and second lanes, a
vps10 strain (EMY3); third and fourth lanes, a
vps10 strain (EMY3) carrying a 2-µm plasmid
containing VTH1 (pEMY106). Panel B, first and second lanes, a
vps10 strain (EMY3)
with a centromere plasmid pSEYC306-PEP4 containing PEP4; third and fourth lanes, a
vps10 strain (EMY3) with a centromere plasmid
pSEYC306-PEP4 containing PEP4 and a 2-µm plasmid
containing VTH1 (pEMY106).
Genetic screens have identified a large number of mutants defective in vacuolar sorting of pro-CPY or of pro-CPY-invertase fusions. The majority of the mutants isolated have pleiotropic defects in vacuolar protein sorting (see (21) for review). However, a few (vps10, vps29, and vps35) turned out to be more specifically defective in the localization of pro-CPY(9, 22) . It has since been shown that one of these genes, VPS10, encodes the receptor that interacts with CPY and sorts this ligand to the vacuole. Deletion of VPS10 results in the missorting of more than 90% of the newly synthesized pro-CPY. However, during normal vegetative growth, less than 10% of PrA or proteinase B is missorted (9) . This observation raised the question of whether an independent sorting mechanism existed for each individual vacuolar protein.
The sorting signal of pro-CPY resides in a sequence (Gln-Arg-Pro-Leu, or QRPL) close to the N terminus of the propeptide(6, 8) . Similarly to pro-CPY, fusion of the PrA propeptide to invertase could direct the fusion protein to the vacuole(10) . Unlike the case of pro-CPY, however, cis-dominant mutations leading to missorting of pro-PrA have not been identified. Indeed, pro-PrA appeared to be sorted correctly to the vacuole even when those parts of the propeptide sufficient for targeting of invertase were exchanged with completely unrelated sequences(12) . In principle, these unrelated sequences could all contain targeting information, although this would seem unlikely. To investigate this problem further, we fused some mutant PrA prosequences, randomized either in the N-terminal or the C-terminal half, to invertase and analyzed the sorting phenotype. Three additional random inserts within each of the two segments were also tested (data not shown), and they showed the same phenotype as those shown in Table 1. This analysis confirmed earlier evidence for propeptide-directed sorting(10) . Thus, PrA-propeptide-invertase fusions that contained sequences unrelated to pro-PrA in place of the C-terminal half of the propeptide were secreted, even though the same mutant propeptides were sorted correctly when fused to the mature region of PrA (i.e., in a normal PrA context). Together these data strongly suggested that PrA can be sorted by two independent signals, one of which is found in the propeptide. The central role for PrA as the primary activating protease for numerous vacuolar proenzymes may explain why there are at least two independent receptors that direct vacuolar delivery of this protease. It furthermore allows for independent regulation of the sorting of CPY and PrA.
The presence of a QRPL-like sequence in one of the
constructs prompted us to test whether Vps10p might be responsible for
the somewhat better sorting of this construct. To our surprise we found
that all of the PrA propeptide-Inv fusion constructs were secreted in a vps10 strain. The observation that Vps10p appeared to be
involved in the sorting of the fusion constructs suggested that it
interacted with, or was involved in the sorting of, native pro-PrA. All
further experiments confirmed this hypothesis.
vps10 strains exhibit a strongly reduced sorting capacity for PrA, and
overproduction of Vps10p is able to suppress the missorting phenotype
associated with moderate overproduction of PrA. Moreover, functional
interaction between pro-CPY and Vps10p would be expected to be subject
to competition by pro-PrA. Indeed, overproduction of pro-PrA leads to
significant secretion of pro-CPY. Importantly, chemical cross-linking
experiments show pro-PrA and Vps10p to directly interact.
QRPL
constitutes the core of the Vps10p recognition sequence in pro-CPY. The
C-terminal half of the PrA propeptide, which confers VPS10-dependent vacuolar sorting of invertase, contains the
similar sequence QKYL. However, this probably is not the signal for
Vps10p-dependent sorting, as the constructs PrA137-rd1-Inv through
PrA137-rd3-Inv, which have a Gln to Glu mutation at this position, are
sorted as efficiently as PrA137-Inv. One possibility is that Vps10p has
more than one binding site and binds the two ligands via different
binding sites. Mutational analysis of the QRPL sequence and its
surroundings in pro-CPY have shown that Vps10p has a fairly broad
specificity(17) . Thus, it may well be that sequences seemingly
unrelated to QRPL can function as ligands for the same binding site on
Vps10p in a way analogous to that by which a peptide interacts with the
binding site of a protease. This would require that they are exposed
and/or in a dynamic/disordered structure. Such extended structures are
likely to be unaffected by fusion to invertase. The notion that many
disordered structures are substrates for Vps10p interaction is
supported by the observation that PrA137pro-Inv, containing only
the 59 N-terminal residues of the mature PrA sequence, is sorted with
approximately 50% efficiency in a VPS10-dependent manner.
Regarding the VPS10-independent sorting signal, it is likely
that it is localized outside the propeptide, since e.g. the
PrA137-Inv fusion is completely missorted in the
vps10 strain. Possible receptor candidates for the VPS10-independent sorting of PrA were Vth1p and Vth2p. These
two proteins are identical in sequence and show close homology to
Vps10p. Vth1p is indeed a vacuolar sorting receptor, as it can suppress
the missorting of PrA and CPY when overproduced in a
vps10 strain. However, the Vth proteins appear to be produced at very
low levels from their normal chromosomal loci. This indicates that the
Vth proteins normally only play a minor role in vacuolar sorting of PrA
and CPY, a conclusion that is supported by the fact that, even though
Vth1p can sort CPY, more than 90% of the newly synthesized pro-CPY
molecules are missorted in a
vps10 strain. Finally, a
strain having a triple deletion of VPS10, VTH1, and VTH2 sorted PrA with the same efficiency as a
vps10 single-deletion mutant. This indicates that yeast contains a
sorting mechanism for PrA that is independent of VPS10, VTH1, and VTH2.
The nature of the VPS10, VTH1, VTH2-independent mechanism of PrA sorting
remains as yet unclear. An interesting recent observation in this
context is that two vps mutants (vps29 and vps35), which sort PrA correctly(9, 22) ,
exhibit a general deficiency in recycling of Vps10p and other
Golgi-located proteins to the Golgi complex. ()This might
suggest that the alternative mechanism of PrA sorting involves a
carrier protein that does not cycle between the late endosome and the
Golgi complex but rather remains trapped in the late endosome or
proceeds to the vacuole. Indeed, PrA may ``piggyback'' a ride
on a normal integral membrane protein of the vacuole. While this
remains speculative at present, a consequence of the redundancy of the
sorting mechanism is that it has been difficult to identify vps mutants affecting pro-PrA specifically. It should be possible to
overcome this problem by employing a
vps10 strain in
future genetic screens.