these degradation assays with PVSE-Yhr020w,
the yeast proline-tRNA synthetase, a long-lived
protein despite its N-terminal Pro (fig. S14C). These
ongoing analyses are far from complete, as the
examined N-terminal Pro–bearing yeast proteins
constitute less than 4% of the full set (figs. S15 to
S17). In sum, while N-terminal Pro–bearing physiological substrates of the S. cerevisiae Pro/N-end
rule pathway other than P-Fbp1, P-Icl1, and P-Mdh2
have not yet been pinpointed, they are likely to exist
and would be expected to be identified through the
approaches described above.
Gid4 and the Pro/N-end rule pathway
We have identified Gid4, a subunit of the
S. cerevisiae GID Ub ligase, as the recognition
component of the GID-mediated proteolytic
system termed the Pro/N-end rule pathway
(Fig. 1B). One function of this system is a rapid
down-regulation of gluconeogenesis, through the
destruction of gluconeogenic enzymes, upon a
return of cells to glucose-replete conditions. Gid4,
the Pro/N-recognin of the Pro/N-end rule pathway, is shown here to target the gluconeogenic
enzymes P-Fbp1, P-Icl1, and P-Mdh2 (and possibly other proteins as well) through the binding
to their N-terminal Pro residues and adjacent sequence motifs (Figs. 1, 3, and 4).
Gid4 was also required for the GID-mediated
degradation of SP-Pck1, the fourth gluconeogenic
enzyme (Fig. 2G and figs. S3B and S11). Degradation of SP-Pck1 involved the recognition of its
internal (position-2) Pro residue (Fig. 2G and
fig. S11). However, in two-hybrid assays, Gid4
did not bind to either full-length SP-Pck1 or its
N-terminal fragment (figs. S4A and S12A). Nonetheless, Gid4 could also be shown to recognize
Pro at position 2, provided that the sequence of
SP-Pck1 proximal to Pro2 was altered by one
residue (figs. S12 and S13). A possible explanation of this result is mentioned above.
The properties of Gid4 discovered so far indicate
that its substrate-binding groove, likely to be
analogous to peptide-binding grooves of antigen-presenting MHC proteins (see above), can recognize either the N-terminal Pro residue or Pro
at position 2 in the presence of cognate adjacent
sequence motifs. The S. cerevisiae genome encodes
~300 proteins that are expected to bear N-terminal
Pro (figs. S15 to S17). Nongluconeogenic substrates
of the Pro/N-end rule pathway that bear N-terminal Pro remain to be identified. The recognition flexibility of the substrate-binding groove of
Gid4 (discussed above; see also Fig. 4 and figs. S12
and S13) suggests that the true diversity of Gid4
substrates is only beginning to be determined.
A notable aspect of GID-mediated processes is
the dichotomy between the GID/proteasome-
mediated degradation of gluconeogenic enzymes
(Fig. 1B) and the “alternative” degradation of the
same enzymes through an autophagy-related
pathway called VID (vacuole import and degrada-
tion) (4–7, 10, 11). If S. cerevisiae is grown on a
nonfermentable carbon source such as ethanol
for less than a day before returning cells to glucose
(i.e., the regimen of the present study), the in-
volvement of VID is negligible (4–7). However,
a much longer growth on a nonfermentable
carbon source results (after return to glucose)
in the VID-mediated degradation of gluconeo-
genic enzymes (8). Remarkably, both proteolytic
processes require the Gid4 Pro/N-recognin of
the present work as well as the other subunits
of the GID Ub ligase (5, 8). It is unclear why
two distinct mechanisms have evolved to con-
ditionally destroy specific proteins bearing Pro/
N-degrons. The design of a circuit for transitions
between the proteasome-based and the VID-based
protein degradation is also unknown. How the
ability of Gid4 to recognize Pro/N-degrons (Fig.
1B) is used by VID (as distinguished from the
proteasome) remains to be understood as well.
Subunits of the S. cerevisiae GID Ub ligase
(Fig. 1, B and C) have sequelogous [similar in
sequence (51)] counterparts in animals and
plants (15–17). Metazoan sequelogs of GID sub-
units form a complex that acts as a Ub ligase
(15, 17). Thus, the discovery that yeast Gid4 is
the Pro/N-recognin (Figs. 1B and 3) will facil-
itate the understanding of the Pro/N-end rule
pathway in other eukaryotes as well.
The Arg/N-end rule pathway (Fig. 1E) does not
recognize N-terminal Pro (22). In addition, the
N-terminal Pro residue is not Nt-acetylated, in
contrast to other N-terminal residues (39, 52).
Consequently, N-terminal Pro cannot confer recognition by the Ac/N-end rule pathway or by the
Arg/N-end rule pathway on a protein bearing this
residue (Fig. 1, D and E). Nonetheless, owing to
the identification of the GID-mediated proteolytic
system as the Pro/N-end rule pathway, all 20 amino
acids of the genetic code have now been shown to
act, in specific sequence contexts, as destabilizing
N-terminal residues (Fig. 1A). Thus, most proteins
in a cell may be conditionally short-lived N-end
rule substrates, either as full-length proteins or as
protease-generated natural protein fragments.
Materials and methods
Yeast strains, media, and
Standard techniques were used for transformation with DNA and for construction of specific
strains (table S1). S. cerevisiae media included
YPD, SD, SE, and SC (see supplementary materials and methods). The alternative carbon
sources, in either liquid or plate media, were
2% ethanol or 2% glucose.
Construction of plasmids
Polymerase chain reaction (PCR) and the Gateway cloning method (Invitrogen) were used for
constructions of plasmids that are cited in table
S2. The oligonucleotide primers are cited in table S3.
The Escherichia coli strains DH5a, SUREII (
Stra-tagene), and STBL2 (Invitrogen) (table S1) were
used for cloning and maintaining plasmids. See
supplementary materials and methods for details.
Most S. cerevisiae protein degradation assays of
this study used a version of the PRT (Fig. 2, A and
B). These reference-based, PRT-based degradation
assays, which used tetracycline-mediated chases,
are described in supplementary materials and
methods. Immunoblotting was carried out using
standard techniques. The results were quantified
using the Odyssey 9120 scanner (Li-Cor).
Two-hybrid and split-ubiquitin
Two initial plasmids for two-hybrid assays were
pGADCg and pGBKCg (table S2). These assays
were carried out largely as described (43–45).
Split-Ub assays (46, 47) for mapping interactions
between Gid4 and (M)P-Fbp1 versus (M)S-Fbp1
involved cotransformations of S. cerevisiae JD52
(table S1) with pCSJ418 (expressing NUb-Gid4-
Flag) and the plasmids pCSJ473 or pCSJ474 (table
S2), which expressed, respectively, (M)P-Fbp1 or
(M)S-Fbp1 linked to the CUb-R-Ura3 moiety. See
supplementary materials and methods for details
of both assays.
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