 |
INTRODUCTION |
A multitude of regulatory circuits, including those that control
the cell cycle, cell differentiation, and responses to stress, involve
metabolically unstable proteins (1-5). A short in vivo half-life of a regulator provides a way to generate its spatial gradients and allows for rapid adjustments of its concentration, or
subunit composition, through changes in the rate of its synthesis or
degradation. Damaged or otherwise abnormal proteins tend to be
short-lived as well (6). Features of proteins that confer metabolic
instability are called degradation signals or degrons (7, 8). The
essential component of one degradation signal, called the N-degron, is
a destabilizing N-terminal residue of a protein (9). A set of amino
acid residues that are destabilizing in a given cell yields a rule,
called the N-end rule, which relates the in vivo half-life
of a protein to the identity of its N-terminal residue. The N-end rule
pathway is present in all organisms examined, from mammals and plants
to fungi and prokaryotes (10, 11).
In eukaryotes, an N-degron consists of two determinants, a
destabilizing N-terminal residue and an internal lysine or lysines (12-15). The Lys residue is the site of formation of a multiubiquitin chain (16). The N-end rule pathway is thus one of the pathways of the
ubiquitin (Ub)1 system. Ub is
a 76-residue protein whose covalent conjugation to other proteins plays
a role in a vast range of biological processes (4, 5, 17). In most of
them, Ub acts through routes that involve the degradation of
ubiquitylated2 proteins by
the 26 S proteasome, an ATP-dependent multisubunit protease (18, 19).
The N-end rule has a hierarchic structure. In the yeast
Saccharomyces cerevisiae, Asn and Gln are tertiary
destabilizing N-terminal residues in that they function through their
deamidation, by the NTA1-encoded3
N-terminal amidase (Nt-amidase), to yield the secondary destabilizing residues Asp and Glu (20). The destabilizing activity of N-terminal Asp
and Glu requires their conjugation, by the ATE1-encoded
Arg-tRNA-protein transferase (R-transferase), to Arg, one of the
primary destabilizing residues (21, 22). The primary destabilizing
N-terminal residues are bound directly by UBR1, also called N-recognin,
the E3 (recognition) component of the N-end rule pathway (10, 11).
In mammals, the deamidation step is mediated by two Nt-amidases,
NtN-amidase and NtQ-amidase, which are
specific, respectively, for N-terminal Asn and Gln (23, 24). In
vertebrates, the set of secondary destabilizing residues contains not
only Asp and Glu but also Cys, which is a stabilizing residue in yeast
(25, 26). The mammalian counterpart of the yeast R-transferase Ate1p
exists as two distinct species, ATE1-1 and ATE1-2, that are produced
through alternative splicing of Ate1 pre-mRNA (27). Both
ATE1-1 and ATE1-2 are similar in specificity to the
ATE1-encoded yeast R-transferase, in that these R-transferases can arginylate N-terminal Asp and Glu, but cannot arginylate N-terminal Cys (27), suggesting the existence of a distinct
R-transferase specific for N-terminal Cys.
UBR1 (N-recognin) of yeast and mammals has two binding sites for the
primary destabilizing N-terminal residues of either proteins or short
peptides. The type 1 site is specific for the basic N-terminal residues
Arg, Lys, and His. The type 2 site is specific for the bulky
hydrophobic N-terminal residues Phe, Leu, Trp, Tyr, and Ile (25, 28,
29). UBR1 contains yet another substrate-binding site, which targets
proteins bearing internal (non-N-terminal) degrons. These proteins
include CUP9 and GPA1 in yeast (30-32) and the encephalomyocarditis
virus 3C protease in metazoans (33).
The known functions of the N-end rule pathway include the control of
peptide import in S. cerevisiae, through the degradation of
CUP9, a transcriptional repressor of the peptide transporter PTR2 (30)
(this control includes a positive feedback mediated by the type 1 and
type 2 sites of UBR1 (31)); the degradation of GPA1, one of two G
proteins in S. cerevisiae (32); and the degradation of
alphaviral RNA polymerases and other viral proteins in infected
metazoan cells (33, 34). Physiological N-end rule substrates were also
identified among the proteins secreted into the cytosol of the
mammalian cell by intracellular parasites such as the bacterium
Listeria monocytogenes (35). Selective perturbation of the
N-end rule pathway was reported to interfere with mammalian cell
differentiation (36, 37) and with limb regeneration in amphibians (38).
Studies of the Ub-dependent proteolysis of endogenous
proteins in muscle extracts suggested that the N-end rule pathway plays
a role in catabolic states that result in muscle atrophy (39).
Until the present work, physiological substrates of Nt-amidases and
R-transferases were unknown in either yeast or larger eukaryotes.
Engineered N-end rule substrates, including the substrates of
Nt-amidases and R-transferases, can be produced in vivo
through the Ub fusion technique, in which a Ub-X-reporter
fusion is cleaved, cotranslationally, after the last residue of Ub by
deubiquitylating enzymes (DUBs) (40), yielding a reporter protein
bearing the predetermined N-terminal residue X (9, 10, 41).
In the present work, we employed a modification of the cDNA-based
sib-selection strategy in a transcription-translation lysate from
rabbit reticulocytes (42, 43) to identify putative physiological substrates of the N-end rule pathway. Specifically, we used dipeptides bearing destabilizing N-terminal residues as selective inhibitors of
the N-end rule pathway, and we screened for mouse cDNAs that expressed proteins whose relative abundance in the lysate was altered
in the presence of relevant dipeptides.
Among the putative N-end rule substrates identified through the use of
this approach was mouse RGS4, a GTPase-activating protein (GAP) for
specific G subunits of heterotrimeric G proteins, and a member of
the family of RGS (regulator of G protein
signaling) proteins (44-50). We discovered that in
addition to the expected removal of N-terminal Met from the newly
formed RGS4, the resulting N-terminal Cys of RGS4 was arginylated,
presumably by a distinct R-transferase whose Cys specificity is
different from that of the known R-transferases. Thus modified RGS4
bore N-terminal Arg, a primary destabilizing residue, and was degraded
by the N-end rule pathway in reticulocyte lysate.
| |
EXPERIMENTAL PROCEDURES |
Plasmids--
The plasmids
pcDNA3-Ub-X-nsP41-254 (where X
is Met, Arg, or Tyr) were used to express
Ub-X-nsP41-254 fusions, denoted below as
Ub-X-nsP4*, from the phage T7 promoter in the transcription-translation reticulocyte lysate system by Promega (Madison, WI). The notation nsP41-254 refers to the
254-residue N-terminal fragment of the 69-kDa nsP4, the Sindbis virus
RNA polymerase (34). The
pcDNA3-Ub-X-nsP41-254 plasmids were constructed using a set of open reading frames (ORFs) encoding fusions
between Ub and the full-length nsP4 (51). The Ub-X-nsP4 ORFs
in pJCEX1 (a gift from Dr. T. Rümenapf, Federal Research Center
for Virus Diseases of Animals, Tübingen, Germany) were used as
templates for PCR with two primers
5'-TTCGGATCCGCCACCATGCAGATCTTCGTGAAGACCCTG-3' and
5'-CCTTCTAGACTATGCGGTGACAAACTCAGTGGTAAT-3', to
produce DNA fragments encoding Ub-X-nsP41-254
fusions (the underlined sequences corresponded to the start and stop
codons, respectively). These fragments were digested with
BamHI and XbaI and cloned into the
BamHI/XbaI-cut pcDNA3 vector (Invitrogen,
Carlsbad, CA).
The plasmid pcDNA3-Ub-Lys-mCL expressed Ub-Lys-mCL, where Lys-mCL
was an N-terminally truncated large subunit of m-calpain starting with Lys-10. The primers
5'-ATTCCGCGGTGGCAAAGACCGCGAGGCGGCCGAGGGGCTG-3' and
5'-CCTACTAGTCTATCATAGGACTGAAAAACTCAGCCACGAGAT-3' were used to
amplify a DNA fragment from pET24-80k, which contained cDNA encoding the large subunit of rat m-calpain (52) (a gift from Dr.
J. S. Elce, Queen's University, Kingston, Ontario, Canada). The
resulting fragment was digested with SacII and
SpeI and cloned into SacII/SpeI-cut
pcDNA3-Ub-Met-nsP41-254. The plasmids pcDNA3-p94
and pcDNA3-p94C129A were used to express the
mouse-specific calpain p94 and its C129A mutant. To produce these
constructs, plasmids containing the rat p94 ORF and the
p94C129A ORF (53) (gifts from Dr. H. Sorimachi and Dr. K. Suzuki, University of Tokyo, Japan) were PCR-amplified and cloned into
the pcDNA3 vector.
The plasmid pcDNA-RGS4 was used for expression of the mouse RGS4 in
reticulocyte lysate and for transfection-mediated expression of RGS4 in
mouse L cells. This plasmid contained the T7 and CMV promoters, and the
mouse RGS4 ORF followed by two stop codons. The RGS4 ORF was
PCR-amplified from the plasmid pcDNA3-26-16-11 (see below),
using the primers
5'-TTCGGATCCGCCACCATGTGCAAAGGACTTGCAGGTCTG-3' and
5'-CCTTCTAGATCATTAGGCACACTGGGAGACCAGGGA-3', followed
by digestion with BamHI and XbaI and cloning into
BamHI/XbaI-cut pcDNA3 (the underlined
sequences corresponded to the start codon and two stop codons,
respectively). The plasmids pcDNA3-RGS4M19A,
pcDNA3-RGS4C2G, pcDNA3-RGS4C2V,
pcDNA3C2A, pcDNA3-RGS4K3R,
pcDNA3-RGS4K3S, and pcDNA3-RGS419-205, which
expressed specific RGS4 mutants, were constructed from pcDNA3-RGS4
using PCR-based site-directed mutagenesis (54).
The plasmid pcDNA3-RGS4M19A-GST expressed
RGS4M19A-GST, a fusion of RGS4M19A and
glutathione transferase (GST). It was constructed through a
PCR-mediated fusion of DNA fragments encoding RGS4 and GST, followed by
cloning into BamHI/XbaI-cut pcDNA3.
PCR-mediated site-directed mutagenesis was then used to introduce the
M19A mutation. The plasmid pcDNA3-RGS16, expressing mouse RGS16
from the T7 promoter (55), and a PCR-produced fragment encoding mouse G
5L were gifts from Dr. C. K. Chen and Dr. M. I. Simon (California Institute of Technology, Pasadena, CA). The entire
coding regions of the final plasmid constructs were verified by DNA sequencing.
In Vitro Transcription-Translation-Degradation System--
The
TNT Quick-coupled Transcription-Translation System (Promega) contained
a rabbit reticulocyte lysate pre-mixed with most of the reaction
components necessary to carry out transcription/translation in the
lysate, including all of the amino acids except methionine. [35S]Methionine (>1,000 Ci/mmol, Amersham Pharmacia
Biotech) was used to label newly formed proteins in the lysate.
Proteins labeled with 3H-amino-acids were produced in the
TNT T7-coupled Reticulocyte Lysate (Promega), a version of the system
where the main components of the reaction were supplied separately. The
reactions were set up according to the manufacturer's instructions.
The reaction mixtures containing dipeptides also contained 0.15 mM bestatin (Sigma), an inhibitor of some aminopeptidases,
to reduce degradation of the added dipeptides (28). Stock samples of
dipeptides were 0.5 M solutions in 10 mM
K-HEPES, pH 7.5. The reactions were incubated at 30 °C, unless
stated otherwise; they were terminated by the addition of SDS-sample
buffer, heated at 95 °C for 5 min, and fractionated by SDS-PAGE,
followed by autoradiography or fluorography. 35S in protein
bands was determined using PhosphorImager (Molecular Dynamics,
Sunnyvale, CA).
Screening of Mouse cDNA Library of Small Pools in Rabbit
Reticulocyte Lysate--
Total RNA was isolated from the brains of
female mice with the guanidine thiocyanate method (54).
Poly(A)+ RNA was purified using Oligotex mRNA kit
(Qiagen, Valencia, CA) and was used to synthesize oligo(dT)-primed
double-stranded cDNA with the SuperScript cDNA synthesis kit
(Life Technologies, Inc.). After ligating the DNA fragments to a
BstXI adapter (Invitrogen, San Diego) and digesting with
NotI, the cDNAs were cloned into BstXI-NotI-cut pcDNA3. The resulting cDNA
library was introduced, by electroporation, into Escherichia
coli SURE cells (Stratagene, La Jolla, CA), and the transformants
were frozen in a number of samples. Titration of the library showed
that it contained 3-4 × 106 independent clones.
Pools of the library cDNAs containing each about 50 clones were
prepared as described (42). Individual pools were added to the T7 TNT
Quick-coupled Transcription-Translation System (Promega) together with
[35S]methionine, in either the presence or the absence of
the mixture of 5 mM Arg--Ala, 5 mM Trp-Ala
and 0.15 mM bestatin. All components of a reaction except
the lysate were gently mixed together, followed by the addition of
lysate. The reactions were performed in the total volume of 6.25 µl
in a 96-well microtiter plate at 30 °C for 3.5 h. These
conditions were chosen after preliminary optimization, using Tyr-nsP4*
as a test protein. Once a cDNA pool was found to express a protein
whose relative abundance was increased in the presence of the
dipeptides, the pool was progressively subdivided and retested, until
the isolation of a single positive cDNA clone. One of the clones
thus isolated, termed pcDNA3-26-16-11, contained as ~3-kilobase
pair mouse RGS4 cDNA (see "Results").
N-terminal Radiochemical
Sequencing--
RGS4M19A-GST was expressed in the TNT
T7-coupled Reticulocyte Lysate System (Promega) in the presence of
either [3H]arginine (49 Ci/mmol),
[3H]leucine (155 Ci/mmol), or [3H]lysine
(87 Ci/mmol) (Amersham Pharmacia Biotech), 1 mM
Arg--Ala, and 0.15 mM bestatin for 30 min at 30 °C,
in the total volume of 0.4 ml. The reaction was stopped by the addition
of 20 mM AMP-PNP (Sigma), a non-hydrolyzable ATP analog,
and the labeled RGS4M19A-GST was partially purified by
affinity chromatography on glutathione-Sepharose 4B (Amersham Pharmacia
Biotech), followed by SDS-10% PAGE and the electrophoretic transfer of
separated proteins in 10% methanol, 10 mM CAPS, pH 11, to
Immobilon-P membrane (Millipore, Burlington, MA). The transferred band
of RGS4M19A-GST was cut out (its position was determined
with respect to stained protein markers) and subjected to multiple
cycles of Edman degradation, using the 476A Applied Biosystems
sequencer (Perkin-Elmer). The fractions from each cycle were collected,
and 3H in the fractions was determined using a
scintillation counter.
Transfections of Mouse L Cells and Pulse-Chase
Assay--
Transient transfections of mouse L cells and pulse-chase
analysis were performed as described previously (56). The
affinity-purified polyclonal antibody raised against a peptide near the
C terminus of RGS4 was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). In the experiments with proteasome inhibitor MG132
(Calbiochem), it was added to cells, at the final concentration of 50 µM, 45 min before the addition of
[35S]methionine and was present throughout the
pulse-chase assay.
| |
RESULTS |
Degradation of N-end Rule Substrates in a Transcription-Translation
System Derived from Reticulocytes--
The N-end rule pathway can be
selectively inhibited in reticulocyte lysates through the addition of
dipeptides bearing either type 1 or type 2 destabilizing N-terminal
residues (25, 28). A commercially produced transcription-translation
system derived from reticulocyte lysate and containing the phage T7 RNA
polymerase was used to express a protein of interest from DNA template
in one reaction mixture. A putative N-end rule substrate in the lysate was detected through its increased concentration in the presence of a
cognate dipeptide inhibitor of the N-end rule pathway. We began by
examining the degradation of model N-end rule substrates, which were
derived from nsP4, the 69-kDa Sindbis virus RNA polymerase, a
physiological substrate of the mammalian N-end rule pathway that bears
N-terminal Tyr (34). The nsP4 protein is produced during Sindbis virus
infection through site-specific cleavage of the viral polyprotein
precursor. In the present work, the Ub fusion technique (9, 10, 51) was
used to synthesize, in reticulocyte lysate, a set of X-nsP4
derivatives that contained the first 254 residues of nsP4 and in
addition bore different residues at the Ub-nsP4 junction. These test
proteins, Ub-Arg-nsP41-254, Ub-Tyr-nsP41-254,
and Ub-Met-nsP41-254, are denoted below as Ub-Arg-nsP4*,
Ub-Tyr-nsP4*, and Ub-Met-nsP4*, respectively. Their N-terminal Ub
moiety was cotranslationally cleaved off by DUBs in the lysate yielding
Tyr-nsP4*, Arg-nsP4*, and Met-nsP4*, respectively. Earlier experiments
by T. Rümenapf have shown that these constructs were N-end rule
substrates in the transcription-translation reticulocyte
lysate.4
When a Ub-X-nsP4* fusion was expressed in reticulocyte
lysate and monitored as a function of time, a major band corresponding to Ub-lacking X-nsP4* was observed (Fig.
1, A and C).
Although the electrophoretic mobility of X-nsP4* proteins
was slightly higher than expected from their predicted molecular mass
of 29 kDa, the observed proteins were clearly X-nsP4*,
rather than, for example, Ub-X-nsP4*, because the removal of
Ub occurs cotranslationally or nearly so (57), and also because
SDS-PAGE of the same samples using a more concentrated polyacrylamide
gel revealed the ~8-kDa band of labeled free Ub (data not shown). In
addition, the degradation patterns of presumed X-nsP4*
derivatives of Ub-X-nsP4* fusions conformed to the N-end
rule, as shown below.
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Fig. 1.
Effects of dipeptides on the accumulation of
Arg-nsP4* and Met-nsP4* in the transcription-translation reticulocyte
lysate. Plasmids expressing Ub-Arg-nsP4* (A) or
Ub-Met-nsP4* (C) were added to reticulocyte lysate in the
presence of [35S]methionine and either in the absence of
N-end rule inhibitors (lane c (controls)) or in
the presence of either 1 mM Arg--Ala (lanes
1), or 1 mM Trp-Ala (lanes 2), or 1 mM Ala-Lys (lanes 3), or 1 mM
Lys-Ala (lanes 4), or 1 mM Ala-Arg (lanes
5). The samples in lanes 1-5 also contained 0.15 mM bestatin. The reactions were carried out for 10, 20, 30, 60, 120, and 240 min, followed by SDS-PAGE, autoradiography, and
quantitation. The position of R-nsP4* band and the multi-Ub-containing
species are indicated on the left. The positions of
molecular mass markers are indicated on the right.
B, relative amounts of 35S-labeled Arg-nsP4* in
A were determined using PhosphorImager and were plotted as a
function of incubation time. The amount of Arg-nsP4* in the reaction
without dipeptides at 30 min was assigned the value of 100. Control
reaction are as follows: in the absence of dipeptides ( ); in the
presence of Arg--Ala ( ); in the presence of Trp-Ala ( ); in the
presence of Ala-Lys ( ); in the presence of Lys-Ala ( ); and in the
presence of Ala-Arg ( ). D, relative amounts of
[35S]Met-nsP4* in C were determined and
plotted as in B.
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The metabolically stable Met-nsP4* accumulated rapidly during the first
30 min of the transcription-translation reaction and reached a plateau
around 30 min, because the protein synthesis (but not the activity of
the N-end rule pathway; see below) ceased in the lysate by that time
(Fig. 1, C and D). Thus, in this setting, the
incubation times in excess of ~30 min were operationally equivalent to the "chase" part of a pulse-chase experiment. In contrast to Met-nsP4*, the relative levels of Arg-nsP4* (Fig. 1, A and
B, lanes labeled c (controls)) and
Tyr-nsP4* (data not shown) began to decrease after 30 min, reflecting
their continuing degradation by the N-end rule pathway. Arg and Tyr
are, respectively, a type 1 (basic) and a type 2 (bulky hydrophobic)
destabilizing residue in the N-end rule (10). A ladder of higher
molecular mass bands, apparently of multiubiquitylated Arg-nsP4*, was
observed with Arg-nsP4* (Fig. 1A) but not with the
metabolically stable Met-nsP4* (data not shown).
The addition of Arg--Ala, a type 1 inhibitor of the N-end rule
pathway (25), strongly inhibited the ubiquitylation and degradation of
Arg-nsP4* (Fig. 1A, lanes labeled 1).
The same dipeptide had no effect on the degradation of Tyr-nsP4*, which bore a type 2 destabilizing N-terminal residue (data not shown). Conversely, Trp-Ala, a type 2 inhibitor, strongly decreased the degradation of Tyr-nsP4* (Fig.
2B) but had little effect on
the degradation of Arg-nsP4* (Fig. 1A, lanes
labeled 2). At 1 mM, none of the several
dipeptides tested, including those bearing type 1, type 2, or type 3 destabilizing N-terminal residues (25), affected the relative band
intensity of Met-nsP4* (Fig. 1, C and D),
indicating that these dipeptides did not perturb transcription and
translation in this system. However, at 10 mM, some of the dipeptides significantly delayed the synthesis of X-nsP4*
(data not shown). Therefore the experiments were carried out with
dipeptides at the initial concentration of 1 mM. At this
concentration, the inhibition of the N-end rule pathway was strong but
incomplete. The inhibition of degradation of, for example, a type 1 substrate such as Arg-nsP4* by a cognate dipeptide could be observed
through large differences in the amount of a test protein produced by 30 min, the time of cessation of protein synthesis in the lysate, and
through even larger differences at 60 min (Fig. 1B).
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Fig. 2.
Lys-mCL is not an N-end rule substrate in
reticulocyte lysate. A, plasmid DNA encoding a
Ub-Lys-mCL, a Ub fusion to a derivative of the large m-calpain subunit
bearing N-terminal Lys (see "Results"), was expressed in the
transcription-translation reticulocyte lysate in the presence of
[35S]methionine. The reaction was allowed to proceed for
20 min at 37 °C ("pulse"). Thereafter, cycloheximide and
unlabeled L-methionine were added to all samples, to the
final concentrations of 0.1 mg/ml and 0.5 mM, respectively.
CaCl2 and/or Arg--Ala were added to some of the samples,
to the final concentrations of 2 and 2.5 mM, respectively.
0.15 mM bestatin was present in these samples as well. The
reactions mixtures were incubated for the indicated chase times of 0, 1, or 2 h, followed by SDS-PAGE and autoradiography. B,
plasmid DNA encoding Ub-Tyr-nsP4* was expressed in the lysate, and the
reactions were allowed to proceed as in A except that
Trp-Ala was added, instead of Arg--Ala, after the pulse. The
positions of (deubiquitylated) Lys-mCL and Tyr-nsP4* bands are as
indicated at the right.
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m-Calpain Bearing N-terminal Lysine Is Not a Substrate of the N-End
Rule Pathway--
m-Calpain is a calcium-activated cysteine protease
composed of an 80-kDa large subunit (mCL) and a 30-kDa small subunit
(mCS) (58). The m- and µ-calpains are ubiquitously expressed in
metazoan cells. In the presence of Ca2+, calpain subunits
undergo autoproteolytic cleavages in their N-terminal regions. The
processed calpains have increased activity. The in vitro
autoproteolytic cleavage of mCL was shown to remove the first 9 residues of mCL, yielding a modified subunit, denoted below as Lys-mCL,
which bears N-terminal Lys (59), a type 1 destabilizing residue (10).
We asked whether Lys-mCL was unstable in reticulocyte lysate and, if
so, whether its instability required N-terminal Lys residue. A plasmid
encoding the Ub-Lys-mCL fusion was constructed, and the fusion was
expressed in the transcription-translation lysate. It was found that
under standard conditions (in the absence of added Ca2+),
the fusion-derived Lys-mCL was metabolically stable in the lysate (Fig.
2A). In the presence of added Ca2+ (2 mM), the newly formed Lys-mCL underwent rapid (apparently autolytic) degradation in the lysate. Significantly, this
Ca2+-induced degradation of Lys-mCL was not inhibited by
Arg--Ala, a type 1 inhibitor of the N-end rule pathway (Fig. 2).
Thus, the Lys-mCL derivative of the large subunit of m-calpain is not a substrate of the N-end rule pathway in reticulocyte lysate, despite the
presence of a destabilizing N-terminal residue (see
"Discussion").
The muscle-specific calpain p94 is homologous to the ubiquitous
calpains and was shown to be metabolically unstable (53, 60). We
expressed rat p94 and its proteolytically inactive mutant p94C129A in reticulocyte lysate. The full-length p94 was
rapidly cleaved in the lysate, yielding several fragments, whereas
proteolytically inactive p94C129A remained stable (data not
shown), in agreement with the earlier evidence (53). Dipeptides
Arg--Ala and Trp-Ala had no effect on the relative yields of bands
corresponding to either the full-length p94 or the products of its
autolysis (data not shown). Thus, neither p94 nor its autolytically
produced fragments are the substrates of the N-end rule pathway.
Degradation of RGS4 in Reticulocyte Lysate Is Decreased by Type 1 Inhibitors of the N-end Rule Pathway and by a Proteasome
Inhibitor--
To search for putative N-end rule substrates in a
systematic way, we employed a modification of the previously developed
in vitro screening method that involves the use of small
pools of cDNA clones (42, 43). A mouse brain cDNA library was
constructed, and about 500 mouse cDNA pools, each containing about
50 cDNAs, were tested, sequentially, in the
transcription-translation reticulocyte lysate. Each reaction was
carried out in either the absence or the presence of a mixture of two
dipeptides, Arg--Ala and Trp-Ala, which have been shown to inhibit
selectively the degradation of engineered N-end rule substrates in the
lysate (Figs. 1, A and B, and 2B). The
resulting 35S-labeled proteins were visualized by SDS-PAGE
and autoradiography. We searched for protein bands that were
selectively enhanced in the presence, but not in the absence, of the
dipeptide inhibitors. A cDNA pool containing a putative substrate
of the N-end rule pathway was subjected to subcloning, followed by
re-analysis in the lysate. This procedure yielded specific cDNAs
encoding putative N-end rule substrates.
Thus far, we found 7 cDNAs that encoded proteins whose expression
patterns identified them as putative substrates of the N-end rule
pathway. Most of these cDNAs encoded N-terminally truncated polypeptides, produced by translation from Met codons that were internal in the corresponding wild type ORFs (data not shown). Although
some of these truncated proteins may prove to be physiologically relevant substrates of the N-end rule pathway, we focused at first on a
putative N-end rule substrate encoded by a full-length cDNA, which
was found to encode the mouse RGS4, a member of the RGS family of GAPs
for specific G subunits of G proteins (44, 45). The ~3-kilobase
pair RGS4 mRNA contained the ~2-kilobase pair long
3'-untranslated region. To optimize the expression of RGS4 in
reticulocyte lysate, we constructed the plasmid pcDNA3-RGS4, which
contained the full-length RGS4 ORF, followed by two stop codons, and lacked the wild type 3'-untranslated region of
RGS4 mRNA. As with the original RGS4 cDNA
isolate (data not shown), the transcription-translation of
pcDNA3-RGS4 yielded two protein bands, of 25 and 22 kDa (Fig.
3, A and D). The
larger protein was the full-length RGS4, whereas the smaller one was
produced by translation that started from a downstream Met (AUG) codon at amino acid position 19, because pcDNA3-RGS4M19A, in
which the Met-19 codon was converted to an Ala codon, did not express
the lower protein band (Fig. 3D).
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Fig. 3.
Type 1 dipeptides
Arg--Ala and Lys-Ala inhibit degradation and
ubiquitylation of RGS4 in reticulocyte lysate. A, the
plasmid pcDNA3-RGS4 (see "Experimental Procedures") was
expressed in the transcription-translation reticulocyte lysate in the
presence of [35S]methionine and either in the absence of
dipeptides (lanes c) or in the presence of 1 mM Arg--Ala (lane 1), 1 mM Trp-Ala (lane 2), 1 mM
Ala-Lys (lane 3), 1 mM Lys-Ala
(lane 4), and 1 mM Ala-Arg
(lane 5). Dipeptide-containing samples also
contained 0.15 mM bestatin. The reactions were carried out
for 10, 20, 30, 60, 120, and 240 min, followed by SDS-PAGE,
autoradiography, and quantitation. The positions of mouse RGS4 and the
multi-Ub-containing species are indicated on the left. An
asterisk marks the N-terminally truncated RGS4 derivatives,
which were produced through alternative translational initiation from
the internal start codon at the Met-19 position of RGS4. The positions
of molecular mass markers are indicated on the right.
B, autoradiographic overexposure of the 20-min part of
B to visualize the ladders of multi-Ub-containing species.
C, relative amounts of 35S-RGS4 in A
were determined using PhosphorImager and were plotted as a function of
incubation time. The amount of RGS4 in the reaction without dipeptides
at 30 min was assigned the value of 100. Control reactions were as
follows: in the absence of dipeptides (); in the presence of
Arg--Ala (); in the presence of Trp-Ala (); in the presence of
Ala-Lys (); in the presence of Lys-Ala (); and in the presence of
Ala-Arg (). D, plasmids encoding either wild type RGS4
(wt), or RGS4M19A, or RGS419-205
were expressed in the lysate either in the absence of dipeptides
(lane c), or in the presence of 1 mM Arg--Ala
and 0.15 mM bestatin (lane 1), or in the
presence of 1 mM Trp-Ala and 0.15 mM bestatin
(lane 2).
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The RGS4 cDNA was initially picked by this screen because upon the
incubation times of 2-4 h, the lower (22-kDa) band increased in
intensity in the presence, but not in the absence, of dipeptide inhibitors. However, this effect was later found to be unrelated to the
N-end rule pathway, as it was observed in the presence of all
dipeptides tested, including those that did not inhibit the pathway
(Fig. 3A). In contrast, at the incubation times from 20 min
to 1 h, the relative amount of the full-length RGS4 increased up
to 5-fold in the presence of either Arg--Ala or Lys-Ala, the type 1 inhibitors of the N-end rule pathway (Fig. 3, A and
C), but not in the presence of Trp-Ala (type 2 inhibitor of
the same pathway), or Ala-Lys and Ala-Arg (type 3 inhibitors) RGS4
(Fig. 4). A ladder of high molecular mass
bands, apparently of multiubiquitylated RGS4, was observed above the
RGS4 and RGS4M19A bands at 10, 20, and 30 min of incubation
(Fig. 3, A and B and data not shown). Arg--Ala
and Lys-Ala, but not the other tested dipeptides, delayed the
appearance of multiubiquitylated RGS4 (Fig. 3, A and
B). The temporal pattern of inhibition of degradation of
RGS4 by dipeptide inhibitors was similar to that described (and
explained) above for the engineered N-end rule substrate Arg-nsP4*
(Fig. 1, A and B). Taken together, these data
suggested that RGS4 was a type 1 substrate of the N-end rule pathway in
reticulocyte lysate.
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Fig. 4.
The relative levels of RGS4 and engineered
N-end rule substrates in the presence of cognate dipeptide inhibitors
in reticulocyte lysate.  , the ratios, denoted as
dipeptides/controls (D/C), of the relative amounts of RGS4
in the presence versus the absence of 1 mM
Arg--Ala, as a function of time in the lysate. These ratios were
calculated from the data in Fig. 3C. , the analogous
ratio curve but for Tyr-nsP4* in the presence of 1 mM
Trp-Ala (primary data not shown). , the analogous ratio curve but
for Arg-nsP4* in the presence of 1 mM Arg--Ala
(calculated from the data in Fig. 1B; the 4-h ratio could
not be determined reliably because of too low 35S in the
band of Arg-nsP4* in the absence of dipeptides). Note the monotonous
increase of the dipeptides/controls ratio for engineered N-end rule
substrate but not for RGS4.
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At longer reaction times (either 2 or 4 h) the effect of type 1 N-end rule inhibitors on the relative level of RGS4 was not observed
(Fig. 3, A and C, and Fig. 4). By contrast, the
band of a model N-end rule substrate such as Arg-nsP4* was stronger in
the presence of Arg--Ala inhibitor at all reaction times, including
2 and 4 h (Fig. 1B and Fig. 4). This difference in the patterns of inhibition of RGS4 and Arg-nsP4* degradation could be
explained if, in contrast to Arg-nsP4*, there were two pools of the
newly formed RGS4 molecules, one of which underwent rapid degradation,
while the other remained long-lived and accumulated during the chase,
after ~30 min of incubation. The fraction of short-lived RGS4 would
be strongly increased by the type 1 inhibitors Arg--Ala or Lys-Ala
at the early times of incubation (before ~1 h), but over longer times
(from 1 to 4 h) this degradation-susceptible form of RGS4 would
still be degraded in the lysate, presumably because of incomplete
inhibition of the N-end rule pathway by dipeptides, in addition to
gradual destruction of the dipeptides by peptidases in the lysate.
Thus, at reaction times of 2 h or longer, only the subset of
long-lived RGS4 molecules remained.
The fraction of short-lived RGS4 comprised 75-80% of the total RGS4;
the remainder of RGS4 molecules (15-25%) was long-lived. This
estimate stemmed from the fact that at 30 min, the amount of RGS4 was 4 to 5 times higher in the presence of Arg--Ala than in its absence
(Fig. 3C, Fig. 4, and data not shown). (That the effect of
Arg--Ala did not result from its influence on the rates of
transcription/translation in the lysate was indicated by control experiments with the long-lived Met-nsP4* (Fig. 1, C and
D).) In contrast to RGS4, most Arg-nsP4* molecules were
short-lived in the lysate. As a result, the relative amount of
Arg-nsP4* was higher in the presence of Arg--Ala than in its absence
throughout the 4-h incubation (Fig. 4), although the amount
of Arg-nsP4* continued to decline after 1 h even in the presence
of Arg--Ala (Fig. 1), for the reasons described above.
We also examined the effect of a proteasome inhibitor, MG115 (61), on
the degradation of RGS4 and Arg-nsP4* in the lysate. MG115 at 10 µM markedly increased the levels of both RGS4 and Arg-nsP4* at the incubation times shorter than 1 h (Fig.
5, A-D). MG115 at 0.1 mM further increased the amounts of RGS4 and Arg-nsP4* at
these incubation times (Fig. 5, A-D). In addition, the
ladders of apparently multiubiquitylated RGS4 and Arg-nsP4* were
enhanced in the presence of MG115 (Fig. 5, A and
C), in contrast to the effect of N-end rule inhibitors,
which decreased the relative level of multi-Ub ladders (Fig.
3B). These patterns were consistent with MG115 inhibiting
post-ubiquitylation steps of the proteasome-mediated degradation of
RGS4 and Arg-nsP4*, in contrast to the action of dipeptides, which
inhibited pre-ubiquitylation steps (25, 61).
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Fig. 5.
Proteasome inhibitor MG115 decreases
degradation of RGS4 and Arg-nsP4* in reticulocyte lysate. RGS4
(A) and Arg-nsP4* (C) were expressed in the
transcription-translation reticulocyte lysate in the presence of
[35S]methionine and either in the absence of MG115
(Me2SO solvent alone; lanes c) or in
the presence of 10 µM (lane 1) and
100 µM (lane 2) MG115. The
reactions were carried out for 10, 20, 30, 60, 120, and 240 min,
followed by SDS-PAGE, autoradiography, and quantitation. The positions
of RGS4 and Arg-nsP4* bands and of the ladder of multi-Ub-containing
species are indicated on the left. B, relative
amounts of 35S-RGS4 in A were determined using
PhosphorImager and were plotted as a function of incubation time. The
amount of RGS4 in the reaction without MG115 at 30 min was assigned the
value of 100. , control reaction, in the absence of MG115; , in
the presence of 10 µM MG115; , in the presence of 100 µM MG115. D, the same as in B but
for Arg-nsP4*.
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Similarly to the effects of dipeptide inhibitors (Fig. 1), the
proteasome level inhibition by MG115 was also incomplete and resulted
in a gradual decrease of the RGS4 and Arg-nsP4* levels after 30 min of
incubation (Fig. 5). Note that this decrease led to the nearly complete
disappearance of Arg-nsP4* by 4 h of incubation, whereas in the
case of RGS4 the decrease stopped when 20-25% of RGS4 still remained
in the lysate (Fig. 5, B and D). These data provided independent evidence for the existence of a
degradation-resistant fraction of RGS4. In summary, we identified mouse
RGS4 as a type 1 substrate of the N-end rule pathway in the
reticulocyte lysate.
The Degradation Signal of RGS4 Is a Cysteine-based
N-degron--
The N-terminally truncated RGS4, produced through
translation initiation at the second (Met-19) start codon of the
RGS4 ORF (see above), was not a substrate of the N-end rule
pathway, because degradation of this RGS419-205 derivative
in the lysate was not selectively decreased by dipeptide inhibitors of
the N-end rule pathway (Fig. 3A and data not shown). This
finding strongly suggested that the UBR1-dependent degron
of the full-length RGS4 was located near its N terminus. The deduced
N-terminal sequence of RGS4 is Met-Cys-Lys-Gly- (62). Extensive
evidence indicates that cytosolic methionine aminopeptidases (MetAPs),
a class of proteases present in all organisms, cleave off N-terminal
Met from proteins and short peptides depending largely on the identity of the next residue, which becomes N-terminal after the cleavage. In
particular, MetAPs rapidly (cotranslationally) cleave Met off N-terminal sequences beginning with Met-Cys (63-65). Cys is a
stabilizing residue in prokaryotes and in the yeast S. cerevisiae but a secondary destabilizing residue in the metazoan
N-end rule pathway (25, 26). Specifically, the destabilizing activity
of N-terminal Cys is tRNA-dependent, strongly suggesting
that an Arg-tRNA-protein transferase (R-transferase) conjugates Arg, a
primary destabilizing residue, to the N-terminal Cys of an N-end rule
substrate (25). This, and the fact that the degradation of RGS4 was
inhibited by type 1 but not by type 2 dipeptides (Fig. 3), suggested
the following model: the N-terminal Met of newly formed RGS4 is removed by MetAPs; the resulting N-terminal Cys is arginylated by
R-transferase, and the arginylated RGS4 is targeted for degradation by
the UBR1-encoded E3 and the rest of the N-end rule pathway.
One prediction of this model was that a significant fraction of RGS4 in
the reticulocyte lysate should contain the N-terminal sequence
Arg-Cys-Lys-Gly-, as distinguished from either Cys-Lys-Gly- or the
encoded sequence Met-Cys-Lys-Gly-. Another prediction was that a
mutational replacement of Cys-2 with a stabilizing residue that still
allows the efficient removal of N-terminal Met should make the
resulting RGS4 variant long- lived in the lysate. We tested both predictions.
Since the conventional N-terminal sequencing of RGS4 produced in the
lysate would have required a major scale up of the reaction protocol,
we employed radiochemical sequencing. A plasmid encoding RGS4M19A fused to glutathione transferase (GST) was
constructed. The RGS4M19A-GST protein was expressed in the
lysate and was found to be degraded by the N-end rule pathway
indistinguishably from the unmodified RGS4 (data not shown). The M19A
mutation was introduced to preclude the formation of N-terminally
truncated RGS4 variant (Fig. 3, A and D).
RGS4M19A-GST was synthesized in the lysate in the presence
of Arg--Ala (type 1 inhibitor of the N-end rule pathway) and either
[3H]arginine, [3H]lysine, or
[3H]leucine. The resulting 3H-labeled
RGS4M19A-GST proteins were purified using
glutathione-Sepharose affinity chromatography and SDS-PAGE. The band of
RGS4M19A-GST was subjected to automated Edman degradation,
and 3H released in each cycle was determined.
RGS4M19A-GST labeled with [3H]arginine
yielded a major peak of 3H in the first Edman cycle,
followed by a second peak in the cycle 14, consistent with Arg at
positions 1 and 14 (Fig. 6A).
Whereas Arg at position 1 would have to be conjugated to RGS4
posttranslationally, as predicted by the above model, the first encoded
Arg residue of RGS4 was, in fact, located at the encoded position 14 of
arginylated RGS4 (in the sequence frame that begins with
Arg-Cys-Lys-Gly-) (Fig. 6A). This experiment was carried out
twice, with two independent preparations of
[3H]RGS4M19A-GST, and gave the same result.
RGS4M19A-GST labeled with [3H]lysine yielded
virtually no at position 1 but produced a major peak at position 3 and elevations at positions 17 and 20, consistent with the known
positions of three Lys residues in the N-terminal sequence of
arginylated RGS4 (Fig. 6B). RGS4M19A-GST labeled
with [3H]leucine yielded no 3H in the cycle 1 but produced peaks in the cycles 5 and 8, once again consistent with
the known positions of two Leu residues in the N-terminal sequence of
arginylated RGS4 (Fig. 6C). Taken together, these
independent sets of radiochemical sequencing data indicated that the
bulk of RGS4 produced in reticulocyte lysate indeed bore the N-terminal
sequence Arg-Cys-Lys-Gly-, with the Arg residue of this sequence not
encoded by RGS4 mRNA.
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Fig. 6.
Radiochemical N-terminal sequencing of RGS4
produced in the reticulocyte lysate. RGS4M19A-GST was
synthesized in the transcription-translation reticulocyte lysate for 30 min in the presence of 1 mM Arg- Ala and one of the
3H-containing amino acids: [3H]arginine
(A), [3H]lysine (B), or
[3H]leucine (C). The resulting
3H-labeled RGS4M19A-GST was purified by
affinity chromatography and SDS-PAGE, followed by radiochemical
N-terminal sequencing through Edman degradation, as described under
"Experimental Procedures." Plotted are the amounts of
3H (cpm) recovered in each Edman cycle. The inferred
N-terminal sequence of RGS4M19A-GST (see "Results") is
shown at the top of each panel, with the expected
3H residues highlighted in bold.
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To verify the second prediction of the model, we constructed several
mutant alleles of RGS4 and expressed them in the lysate in either the
absence or presence of N-end rule inhibitors (Fig. 7). It was found that the replacement of
Cys-2 with either Gly, Val, or Ala residues completely stabilized the
resulting RGS4C2G, RGSC2V, and
RGS4C2A in the lysate (Fig. 7), in agreement with the
model's prediction. We also converted Lys-3 of RGS4 into either Ser or
Arg. Interestingly, whereas the Lys 
Ser replacement completely stabilized the resulting RGS4K3S variant, the Lys Arg
replacement had no effect on the degradation of RGS4K3R
(Fig. 7). Thus, although Lys-3 is not required for ubiquitylation of
RGS4 (since it could be replaced with the non-ubiquitylatable Arg), the
presence of a basic residue (either Lys or Arg) at position 3 (position
2 after the removal of N-terminal Met) is required for the RGS4 degradation by the N-end rule pathway.
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Fig. 7.
Single residue substitutions at the N
terminus of RGS4 abolish its degradation in reticulocyte lysate.
Wild type mouse RGS4 and the indicated single residue derivatives of
RGS4p were expressed in the transcription-translation reticulocyte
lysate in the presence of [35S]methionine and either in
the absence of N-end rule inhibitors (lane c), or
in the presence of 1 mM Arg--Ala (lane
2), or in the presence of 1 mM Trp-Ala
(lane 2). Dipeptide-containing samples also contained 0.15 mM bestatin. The reactions were carried out for 15, 30, 60, and 120 min, followed by SDS-PAGE and autoradiography.
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The fact that RGS4K3S, which bears N-terminal Cys and
differs from RGS4 exclusively at position 3 (Lys Ser replacement), was stable in the lysate (Fig. 7) indicated that the presence of
N-terminal Cys was not sufficient for making a protein an N-end rule
substrate. To explore this issue with other natural proteins, we
expressed in the lysate the mouse protein G5L, a member
of the family of subunits of heterotrimeric G proteins (66). The
predicted N-terminal sequence of G5L is
(Met)-Cys-Asp-Gln-Thr-. We found that Arg--Ala, the type 1 inhibitor
of the N-end rule pathway, had no effect on the pattern of accumulation
of G5L in reticulocyte lysate (data not shown).
The encoded N-terminal sequence of RGS4 is similar to those of RGS5 and
RGS16; the similarities include Cys-2 and a basic residue at position 3 (55, 67). To test whether RGS16 was an N-end rule substrate, we
expressed RGS16 in reticulocyte lysate. As shown in Fig.
8A, the relative amount of
RGS16 was indeed significantly higher in the presence of Arg--Ala
(type 1 inhibitor) than either in the presence of Trp-Ala (type 2 inhibitor) or in the absence of dipeptides. The temporal pattern of the
inhibitor-produced increase in the RGS16 concentration was similar to
that described for RGS4 in Fig. 3B (note the 30- and 60-min
points in Fig. 8A). The effect of Arg--Ala on the
accumulation of RGS16 (Fig. 8A), although significant, was
smaller than its effect on the accumulation of RGS4 (Fig.
3A). Longer autoradiographic exposures showed the ladders of
multi-Ub chains, apparently conjugated to RGS16 (Fig. 8B).
Consistent with the weaker effect of Arg--Ala, the relative level of
RGS16-specific multi-Ub chains (in the absence of inhibitors) was lower
than that of RGS4-specific multi-Ub chains (Figs. 3A and
8B and data not shown). Finally, the ubiquitylation of RGS16 was significantly delayed in the presence of Arg--Ala (type 1 inhibitor) but not in the presence of Trp-Ala (type 2 inhibitor) (Fig.
8B). Taken together, these data (Fig. 8) indicated that RGS16 was also a substrate of the N-end rule pathway in reticulocyte lysate.
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Fig. 8.
Type 1 N-end rule inhibitor decreases
degradation and ubiquitylation of RGS16 in reticulocyte lysate.
A, RGS16 was expressed in the transcription-translation
reticulocyte lysate in the presence of [3H]leucine and
either in the absence of N-end rule inhibitors (lane
c) or in the presence of 1 mM Arg--Ala
(lane 1) or 1 mM Trp-Ala (lane
2). Dipeptide-containing samples also contained 0.15 mM bestatin. The reactions were carried out for 10, 20, 30, 60, 120, and 240 min, followed by SDS-PAGE and fluorography.
B, the same as in A, but with autoradiographic
overexposure to visualize the ladders of multi-Ub-containing
species.
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RGS4 Is Metabolically Unstable in Vivo--
A mouse
RGS4-expressing plasmid was transiently transfected into mouse L cells,
and the metabolic stability of RGS4 was measured in a pulse-chase
assay, using an antibody to RGS4. The transiently expressed RGS4 was
found to be short-lived in L cells, with the half-life of 40-50 min
(Fig. 9, A and B).
When L cells were treated with the proteasome inhibitor MG132, both
before and during pulse-chase, RGS4 was significantly stabilized (Fig.
9, A and B). Thus, similarly to the results with
reticulocyte lysate, the in vivo degradation of RGS4 was
proteasome-dependent.
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Fig. 9.
In vivo degradation of RGS4 in
transiently transfected mouse L cells. A, L cells were
transfected with an RGS4-expressing plasmid (see "Experimental
Procedures"). Cells were preincubated in methionine-free medium for
45 min and then labeled for 12 min with
[35S]methionine/cysteine and chased for 0, 1, or 2 h. Either 50 µM MG132 (from a stock solution in
Me2SO) or the equivalent amount of Me2SO were
present during both preincubation in methionine-free medium and the
pulse-chase. Cell extracts were immunoprecipitated with anti-RGS4
antibody, followed by SDS-PAGE and autoradiography. B, the
pattern in A was quantitated using PhosphorImager. The
amount of 35S in RGS4 at time 0 (end of the pulse) was
taken as 100%. , no proteasome inhibitor; circo, in the presence of
50 µM MG132.
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To verify whether the degradation of RGS4 in vivo required
its Cys-based N-degron, L cells were transfected with the plasmids expressing the RGS4C2G and RGS4C2V mutants, and
the in vivo decay curves of these proteins were compared
with that of wild type RGS4. It was found that these variants of RGS4,
which were completely stable in reticulocyte lysate (Fig. 7), were
degraded in L cells similarly to wild type RGS4 (data not shown).
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DISCUSSION |
We searched for substrates of the mammalian N-end rule pathway by
testing some of the previously known candidates and also by employing a
modification of the sib selection-based in vitro screen (42,
43) in a transcription-translation reticulocyte lysate. Two N-end rule
substrates thus identified were mouse RGS4 and RGS16. These proteins
are members of the RGS family of GTPase-activating proteins (GAPs) that
down-regulate specific G proteins (44-50, 67). We report the following results.
1) The in vitro activated, proteolytically processed larger
(80 kDa) subunit of the cysteine protease m-calpain was previously shown to bear N-terminal Lys (59), a type 1-destabilizing residue in
the N-end rule (10). We expressed this subunit, termed Lys-mCL, in the
reticulocyte lysate as a Ub-Lys-mCL fusion. The resulting Lys-mCL was
not a substrate of the N-end rule pathway, despite the presence of a
destabilizing N-terminal residue. Since an N-degron is a bipartite
signal (12, 13, 15), the absence of active N-degron from Lys-mCL could
be due to the absence of a targetable internal Lys residue, the second
determinant of an N-degron. Another possibility is a steric hindrance
in the binding of UBR1 to the N-terminal lysine of Lys-mCL. Thus, a
destabilizing N-terminal residue is not the sole essential determinant
of an active N-degron, as demonstrated previously with engineered N-end
rule substrates (12).
2) A major fraction (75-85%) of mouse RGS4 synthesized in
reticulocyte lysate was rapidly degraded by a pathway that was
proteasome-dependent and apparently also
Ub-dependent. The other 15-25% of the newly made RGS4 was
found to be stable in the lysate.
3) The degradation of RGS4 was inhibited by dipeptides bearing type
1-destabilizing N-terminal residues (Arg--Ala or Lys-Ala) but was
unaffected by dipeptides bearing a type 2-destabilizing N-terminal
residue (Trp-Ala) or a type 3 residue (Ala-Lys and Ala-Arg).
4) Radiochemical sequencing of RGS4 produced in reticulocyte lysate and
labeled with either [3H]arginine,
[3H]lysine, or [3H]leucine revealed the
presence of posttranslationally conjugated Arg at the N terminus of
RGS4. The deduced N-terminal sequence of RGS4 was Met-Cys-Lys-Gly-. The
observed/inferred sequence was Arg-Cys-Lys-Gly-. This result and,
independently, the fact that degradation of RGS4 was inhibited by type
1 but not by type 2 dipeptides strongly suggested the following model:
the N-terminal Met of newly formed RGS4 is removed by MetAPs; the
resulting N-terminal Cys is arginylated by R-transferase; and the
arginylated RGS4 is targeted for processive degradation by the
UBR1-encoded E3 and the rest of the N-end rule pathway.
5) In agreement with a prediction of this model, the degradation of
RGS4 in reticulocyte lysate was found to require Cys-2 residue, which
becomes N-terminal after the MetAP-mediated cotranslational removal of
Met. Specifically, the RGS4 mutants C2G, C2V, and C2A, in which Cys-2
was replaced with Gly, Val or Ala, were completely stable in the
lysate. The former two residues are stabilizing in the N-end rule, and
neither of the 3 residues is expected to interfere with the removal of
N-terminal Met by MetAPs (64).
6) A Lys Ser replacement at the encoded position 3 of RGS4 also
completely stabilized the resulting RGS4K3S protein.
However, a Lys Arg replacement at this position had no effect on
the degradation of RGS4K3R. Thus, Lys-3 is not required for
ubiquitylation of RGS4. However, the presence of a basic residue
(either Lys or Arg) immediately after Cys is essential for the RGS4
degradation by the N-end rule pathway. One possibility is that the
requirement for a basic residue at this position reflects the substrate
specificity of an uncharacterized R-transferase that arginylates the
N-terminal Cys.
7) The mouse protein G5L, a member of the family of subunits of heterotrimeric G proteins (66) that bore the (initial) N-terminal Met-Cys, was tested and found not to be an N-end rule substrate, similarly to the RGS4K3S mutant, which also bore
the (initial) N-terminal sequence Met-Cys but was not degraded by the
N-end rule pathway. Thus, the N-terminal Cys of wild type RGS4 is an
essential determinant of its N-degron, but other N terminus-proximal residues are relevant as well.
8) Similarities among the N-terminal sequences of RGS4, RGS5, and RGS16
(67) suggested that RGS5 and RGS16 may also be N-end rule substrates.
This prediction was tested, thus far, with RGS16, and was confirmed.
9) RGS4 was transiently expressed in mouse L cells and found to be an
unstable protein, with the half-life of 40-50 min. The in
vivo degradation of RGS4 was proteasome-dependent, as
indicated by the nearly complete stabilization of RGS4 in the presence
of proteasome inhibitor MG132. However, the targeting of RGS4 in this
in vivo setting involved primarily a degron distinct from the Cys-based N-degron, because N-terminal variants of RGS4 such as
RGS4C2V and RGS4C2G, which were completely
stable in reticulocyte lysate, remained unstable in L cells.
We consider the latter result first. 15-25% of the reticulocyte
lysate-produced RGS4 was resistant to degradation by the N-end rule
pathway (paragraph 2 above). An analogous protection of RGS4 against
targeting by the N-end rule pathway in L cells might involve a much
higher fraction of the newly formed RGS4. The mechanism of protection
may be a modification of the N-terminal Cys, for example, its
palmitoylation (45, 47, 62, 67) or acetylation (68) that would be
expected to preclude the arginylation and degradation of RGS4 by the
N-end rule pathway. Thus it is possible, indeed likely, that there is a
kinetic competition among these reactions at the N-terminal Cys of a
nascent RGS4 protein. In experiments to address this model, we added
varying amounts of acetyl-CoA or palmitoyl-CoA (the substrates of
N-terminal acetylases and palmitoyltransferases) to reticulocyte
lysate; no effect of the added compounds on the degradation of newly
formed RGS4 in the lysate was
observed.5 A second
possibility is a spatial localization of RGS4 in L cells that protects
it from degradation by the N-end rule pathway but leaves RGS4 still
targetable by another proteasome-dependent pathway(s) that
is inactive in reticulocytes. Yet another possible reason for the
difference between the results with reticulocyte lysate versus L cells is that the cysteine branch of the N-end rule
pathway may be cell type-specific; for example, a Cys-specific
R-transferase is present in reticulocytes but might be expressed at a
lower level in L cells. In recent experiments, RGS4 was expressed in Xenopus laevis oocytes, through microinjection of
RGS4 mRNA. It was found that, similarly to the results
with reticulocyte extracts, RGS4 was degraded in oocytes by the
cysteine branch of the N-end rule
pathway.6
One difficulty in identifying physiological N-end rule substrates (as
distinguished from those produced through the Ub fusion technique (10))
is caused by the fact that MetAPs remove N-terminal Met from newly
formed proteins if, and only if, the second residue in a polypeptide
chain is stabilizing in the yeast-type N-end rule. Specifically, the
known MetAPs remove N-terminal Met if the second position residues are
Gly, Val, Ala, Ser, Thr, Cys, or Pro (63-65). All of these residues
are stabilizing in the yeast-type N-end rule (10). Therefore, a natural
substrate of an N-end end rule pathway that bears an N-degron in, for
instance, S. cerevisiae cells can be produced exclusively
through cleavages (anywhere along the polypeptide chain) by proteases
distinct from MetAPs. An example of N-end rule substrate of this class
is the S. cerevisiae protein SCC1, a subunit of the cohesin
complex that holds together sister chromatids. In a reaction that
requires ESP1, the 566-residue SCC1 is cleaved (at the time of sister
chromatid separation) at positions 180 and/or 268, resulting in
fragments that bear N-terminal Arg, a type 1-destabilizing residue
(69). These fragments were found to be degraded by the N-end rule
pathway in vivo.7
The situation in metazoans such as mammals is similar to the one in
yeast, in that the yeast-type destabilizing residues cannot be exposed
at the N termini of mammalian proteins through cleavages by the known
MetAPs (10, 64). The difference here is that Cys, Ala, Ser, Thr, and
Pro are stabilizing residues in yeast but destabilizing in mammalian
cells (10, 25, 70). In contrast to the other destabilizing residues,
the second position Cys, Ala, Ser, Thr, and Pro can be efficiently
exposed at the N termini of newly formed proteins through the action of
MetAPs. Of these five residues, N-terminal Cys is a special case; its
destabilizing activity requires its posttranslational conjugation to
Arg, a primary destabilizing residue recognized by the type 1 site of UBR1 (E3) (10).
The finding of the dependence of destabilizing activity of N-terminal
Cys on the presence of tRNA (25, 26), and the identification, in the
present work, of Arg as the posttranslationally linked N-terminal
residue of mouse RGS4, indicated the existence of an Arg-tRNA-protein
transferase (R-transferase) that conjugates Arg to N-terminal Cys. This
R-transferase is distinct from the two previously characterized
mammalian R-transferases, both of which are encoded by the
alternatively spliced ATE1 gene (27). The two forms of mouse
ATE1 R-transferase have different specific activities but apparently
the same substrate specificity; similarly to their S. cerevisiae homolog, they arginylate N-terminal Asp and Glu but
cannot arginylate N-terminal Cys (27).
The ~20 known proteins of the mammalian RGS family have in common the
~130-residue RGS domain that binds to G subunits and is
responsible for the GAP function of RGS proteins (44, 45). As would be
expected of pleiotropic regulators at key junctions of the cellular
metabolism, the activity of RGS proteins is controlled at several
levels, including regulation of their synthesis and localization (44).
It is becoming increasingly clear that the (conditional) degradation of
RGSs is yet another way in which the activity of these proteins is
modulated in cells. Besides RGS4 and RGS16 of the present work, one
other member of the family, RGS7, was recently shown to be unstable
in vivo (71, 72). The normally short-lived RGS7 is
stabilized through its interaction with polycystin, a
PKD1-encoded protein involved in polycystic kidney disease
(71). The degradation of RGS7 is also decreased upon exposure of cells
to tumor necrosis factor ; the resulting increase in the
concentration of RGS7 may contribute to sepsis-induced changes in the
nervous system (72). The degradation signal(s) of RGS7 remains to be characterized.
Our major results are the findings that RGS4 and RGS16 (and possibly
also RGS5) are substrates of the cysteine branch of the N-end rule
pathway and that the Cys-based N-degron of these proteins functions
through the posttranslational arginylation of N-terminal Cys by an
unidentified, apparently Cys-specific R-transferase. RGS4 and RGS16 are
the first physiological substrates of the N-end rule pathway that bear
a secondary destabilizing N-terminal residue, which functions through
its conjugation to Arg, a primary destabilizing residue.
Specific functions of the metabolic instability of RGS4 and RGS16
remain to be understood. One route to these functions is through the
cloning of Cys-specific R-transferase and construction of mouse strains
that lack this enzyme and (therefore) the cysteine branch of the N-end
rule pathway. It would also be important to define, in detail,
Cys-proximal N-terminal sequences in proteins that promote the
arginylation of N-terminal Cys. This information will facilitate the
identification of Cys-specific N-end rule substrates in data bases of
protein sequences.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Division of Biology,
147-75, California Institute of Technology, 1200 E. California Blvd.,
Pasadena, CA 91125. Tel.: 626-395-3785; Fax: 626-440-9821; E-mail:
avarsh@caltech.edu.
