Whereas Kaptinov & Yu [3] suggested that the second of the two Glu residues in the EX7E motif may serve as a nucleophile, and the first as an acid base catalyst, the results of site-direc
Trang 1Comparative importance in vivo of conserved glutamate residues
the UDP-GlcNAc-binding subunit of the first enzyme
in glycosylphosphatidylinositol assembly
Zlatka Kostova1, Benjamin C Yan1, Saulius Vainauskas2, Roberta Schwartz2, Anant K Menon2
and Peter Orlean1
1 Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA; 2 Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
Saccharomyces cerevisiaeGpi3p is the
UDP-GlcNAc-bind-ing and presumed catalytic subunit of the enzyme that forms
GlcNAc-phosphatidylinositol in
glycosylphosphatidylinosi-tol biosynthesis It is an essential protein with an EX7E motif
that is conserved in four families of retaining
glycosyl-transferases All Gpi3ps contain a cysteine residue four
residues C-terminal to EX7E To test their importance for
Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of
S cerevisiaeGpi3p, as well as Cys301, were altered by
site-specific mutagenesis, and the mutant proteins tested for their
ability to complement nonviable GPI3-deleted haploids
Gpi3p-C301A supported growth but membranes from
C301A-expressing cells had low in vitro
N-acetylglucosami-nylphosphatidylinositol (GlcNAc-PI) synthetic activity
Haploids harboring Gpi3p-E289A proved viable, although
slow growing but Gpi3-E297A did not support growth The
E289D and E297D mutants both supported growth at
25C, but, whereas the E289D strain grew at 37 C, the E297D mutant did not Membranes from E289D mutants had severely reduced in vitro GlcNAc-PIsynthetic activity and E297D membranes had none The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37C, but the E285D mutant was nonviable Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps Keywords: endoplasmic reticulum; glycosylphosphatidyl-inositol; glycosyltransferase; Saccharomyces cerevisiae; Schizosaccharomyces pombe
Glycosyltransferases can be classified into a range of
families based on amino-acid sequence similarities, and
these sequence alignments have led to the identification of
signature motifs of amino acids [1–3] Members of the large
Pfam GT1F glycosyltransferase family, with representatives
in the bacteria, archaea, and eukaryotes, have in turn been
classified into subfamilies Many Pfam GT1F
glycosyl-transferases fall into family 4 of the classification proposed
by Campbell and coworkers (CaZY) [2], and some into
families 3 and 5 Most of these retaining glycosyltransferases
have the signature motif EX7E The conservation of these
two acidic residues strongly suggests that they have key roles
in glycosyltransferase activity, and this has been
demon-strated in site-specific mutagenesis and in vitro activity assay studies of CaZY family 3 human muscle glycogen synthase and the family 4 a-mannosyltransferase from Acetobacter xylinum(AceA) [4,5]
The two glutamate residues have been proposed to be involved in catalysis, but their contributions have yet to be evaluated by 3D structural analysis or identification of enzyme–substrate complexes or reaction intermediates Whereas Kaptinov & Yu [3] suggested that the second of the two Glu residues in the EX7E motif may serve as a nucleophile, and the first as an acid base catalyst, the results
of site-directed mutagenesis studies of human muscle glycogen synthase and AceA indicated that, in both cases, mutations of the first glutamate had more severe effects on
in vitro enzyme activity and on the ability of the mutant enzyme to catalyze its glycosyltransfer reaction when expressed in a heterologous system These findings indicated that the first of the Glu residues is critical for enzyme activity, possibly as the nucleophile [4,5]
We showed recently that a yeast EX7E motif protein, Gpi3p, binds a photoactivatable sugar nucleotide analogue, consistent with its function as the substrate-binding and catalytic subunit of the enzyme complex that forms N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) in the first reaction of the pathway for
glycosylphosphatidyl-Correspondence toP Orlean, Department of Microbiology, University
of Illinois at Urbana-Champaign, 309 Roger Adams Laboratory,
600 South Mathews Avenue, Urbana, IL 61801, USA.
Fax: + 1 217 244 5858, Tel.: + 1 217 333 4139,
E-mail: p-orlean@uiuc.edu
Abbreviations: AceA, a-mannosyltransferase from Acetobacter
xylinum; GPI, glycosylphosphatidylinositol; GlcNAc-PI,
N-acetylglucosaminylphosphatidylinositol.
(Received 26 July 2003, revised 11 September 2003,
accepted 19 September 2003)
Trang 2inositol (GPI) biosynthesis [6] As Gpi3p is probably a
glycosyltransferase, and because it is encoded by an essential
gene in both Saccharomyces cerevisiae and, as we report
here, Schizosaccharomyces pombe, it presents an excellent
model with which to assess the effects of amino-acid
replacements in the EX7E motif by testing mutated forms of
Gpi3p for their ability to complement lethal null mutations
in GPI3 The results of our studies indicate that both Glu
residues are important for function, but that the second Glu
in the EX7E motif is less tolerant of changes to other amino
acids, and therefore comparatively more important for
enzyme function
Gpi3 proteins from various organisms also have a
conserved cysteine (Cys301 in S cerevisiae Gpi3p) On
account of its proximity to the proposed catalytic EX7E
motif, and because GlcNAc-PIsynthetic activity can be
inhibited irreversibly by agents that alkylate thiol groups but
protected from inhibition by uridine nucleotide compounds
[7], it has been speculated that this cysteine is important for
function, perhaps for UDP-GlcNAc binding [7,8]
Alter-ation of Cys301 to Ala has no obvious effect on the mutant
protein’s ability to support cell growth, but membranes
harboring the mutant protein had significantly lower GPI
GlcNAc transferase activity than wild-type membranes
The results of our in vivo tests for Gpi3p function do not
support previous suggestions that the first Glu of an EX7E
glycosyltransferase is the nucleophile in the reactions
cata-lyzed by members of this protein family or that Cys301 is
involved in UDP-GlcNAc binding by Gpi3 proteins
Materials and methods
Materials
UDP-[U-14C]GlcNAc (specific radioactivity, 283 mCiÆ
mmol)1) was purchased from NEN Life Science Products
(Boston, MA, USA) Palmitoyl-CoA and tunicamycin were
obtained from Sigma, and Nikkomycin-Z from
Calbio-chem Silica gel 60 TLC plates were supplied by Altech
(Deerfield, IL, USA) X-OMAT X-ray film and
Transcreen-LE intensifying screens were from Eastman-Kodak
Com-pany (Rochester, NY, USA) Expand High Fidelity PCR
mix, Pwo polymerase and EDTA-free protease inhibitor
tablets were purchased from Roche Diagnostics
(Indiana-polis, IN, USA) Geneticin (G418), Taq polymerase, and the
restriction endonucleases were obtained from Gibco-BRL
(now Invitrogen, Carlsbad, CA, USA), and DpnIfrom
Stratagene (La Jolla, CA, USA) Oligonucleotides were
synthesized by Integrated DNA Technologies (Coralville,
IA, USA), and DNA sequencing was performed at the
University of Illinois Genetic Engineering and Sequencing
Facility or at the University of Wisconsin-Madison
Bio-technology Center
Yeast strains and culture media
The temperature-sensitive S cerevisiae gpi3-15C strain had
the genotype MAT a, ade2, leu2-3,112, trp1-1, his3-11,15 [9]
Diploid strain YMW3 (MATa/a, ade2-1/ade2-1, ade3D22/
ade3D22, his3-11,15/his3-11,15, leu2-3,112/leu2-3,112,
trp1-1/trp1-1, ura3-1/ura3-1, can1-100/can1-100) is described
in reference [10], and construction of the heterozygous
GPI3/gpi3::kanMX4 diploid derived from YMW3 is detailed in reference [6] Schizosaccharomyces pombe strains were derived from the wild-type heterothallic strains KGY246 (h–, ade6-M210, ura4-d18, leu1-32) and KGY249 (h+, ade6-M216, ura4-d18, leu1-32)
YPD and SD media were prepared as described in reference [11], and EMM2 medium is described in reference [12] The presence of the kanMX4 marker was verified by scoring for resistance to 200 lg G418ÆmL)1on solid YPD medium
Expression and mutagenesis ofS cerevisiae GPI3
A 2624-bp XhoI–SacIfragment of S cerevisiae genomic DNA, which contained the GPI3 gene, an additional 674 bp DNA containing the native GPI3 promoter at the gene’s 5¢ end, and 494 bp 3¢ flanking DNA, was cloned into the centromeric and 2l plasmids pRS415 and pRS425 [13] The resulting plasmids, pRS415-GPI3 and pRS425-GPI3, were used as templates for mutagenesis and for expression of the GPI3mutants in S cerevisiae
The following mutations were made: E289A, E297A, E289D, E297D, E289G, E297G, E289D/E297D and C301A Mutagenesis of GPI3 was performed using the Stratagene QuikChange site-directed mutagenesis strategy For each mutation to be introduced, a mutagenic oligo-nucleotide and its inverse complement were designed that introduced the appropriate nucleotide changes and a diagnostic restriction site in the middle of the oligonucleo-tides DNA amplification by PCR was carried out using Pwo polymerase Potential mutagenized plasmids were identified by digestion with a restriction enzyme specific for the introduced site, and the GPI3 region on selected plasmids was sequenced to verify the presence of the desired mutation, and the absence of mutations introducing any further amino-acid changes To make the E289D/E297D double mutant, 2l plasmids with each single mutation were mutagenized a second time using the oligonucleotide pairs designed to introduce the additional mutation Double mutants were obtained with each starting mutant plasmid, and the correctness of the mutations and GPI3 sequence was confirmed by DNA sequencing
Cloning, disruption and site-directed mutagenesis
ofSz pombe gpi3+
A BLAST search [14] using the amino-acid sequence of
S cerevisiae Gpi3p as query identified Sz pombe ORF SPBC3D5 This putative Sz pombe gpi3+ gene contains four introns, and sequencing of a gpi3+cDNA amplified from an Sz pombe cDNA library [15] confirmed that the four introns are spliced as predicted
To disrupt the gpi3+gene, DNA fragments of 1 kb each of chromosomal DNA that flanks the 5¢ and 3¢ ends of the gpi3+ locus were amplified by PCR, and the PCR-amplified Sz pombe ura4+ gene was cloned between the two gpi3+-flanking fragments The resulting 3.5-kb frag-ment, in which 88% of the gpi3+sequence was replaced by ura4+ DNA, was used to transform an adenine-proto-trophic diploid created by mating haploid strains KGY246 and KGY249 to uracil prototrophy Stable diploids were selected, and the presence of the disrupting fragment at the
Trang 3chromosomal gpi3+ locus verified by whole-cell PCR.
Diploids were allowed to sporulate, and random spore
analysis was carried out on EMM medium supplemented
with limiting adenine but selective for uracil prototrophy to
identify potential gpi3+::ura4+haploids Tetrad analysis
was also carried out on asci derived from two independent
gpi3+/gpi3+::ura4+ diploids, and viable ade– haploid
segregants were scored for uracil prototrophy
Genomic Sz pombe DNA consisting of the gpi3+locus
and about 700 bp 5¢ flanking DNA and 1000 bp 3¢ flanking
DNA was cloned into the LEU2-marked Sz pombe
expression vector pSP1 [16] Diploids transformed with this
plasmid, pSP1-gpi3+, yielded ade–, uracil and leucine
prototrophic haploids upon sporulation, indicating that
the cloned gpi3+ gene complemented the gpi3+::ura4+
disruption Plasmid pSP1-gpi3+was used as template for
site-directed mutagenesis of Glu277 and Glu285 to Asp as
detailed for S cerevisiae Gpi3p above, and the presence
of the desired mutation, and the absence of mutations
introducing any further amino-acid changes, were verified
by DNA sequencing
Assay of GlcNAc-PI synthesis
Washed mixed membranes were prepared and assayed for
in vitroGlcNAc-PIsynthetic activity as described previously
[6, 17] In assays to estimate the formation of [14
C]GlcNAc-PIwith time, palmitoyl-CoA was omitted from the
incuba-tion mixtures Radiolabeled lipids were extracted, separated
by TLC, and detected by fluorography The
chromato-grams were scanned by Phosphorimager to determine the
relative amounts of14C in the GlcNAc-PIin each sample
Imaging and microscopy
Images of yeast growth on solid YPD medium were
obtained using a Bio-Rad Gel-Doc2000 Growth of
individual colonies arising from spores that had germinated
on solid YPD medium was monitored using a Nikon TE300
inverted microscope with a 40· bright field objective
Results
The importance of the conserved Glu residues in the EX7E
motif of S cerevisiae Gpi3p for in vivo function was tested
by introducing mutations into the GPI3 gene that altered
these residues, Glu289 and Glu297, to aspartates, glycines
or alanines The mutated genes were in turn introduced into
a heterozygous GPI3/gpi3::kanMX4 diploid on low or high
copy plasmids, the diploids induced to undergo meiosis and
sporulation, and the resulting asci dissected to assess
whether the mutated GPI3 gene permitted growth of
otherwise nonviable haploid gpi3::kanMX4 segregants
The consequences of changing the Glu residues in the
EX7E motif of Sz pombe Gpi3p to Asp residues were
exam-ined analogously The C301A mutation in S cerevisiae
Gpi3p was also tested
E289A and E297A mutants inS cerevisiae Gpi3p
Tetrads arising from GPI3/gpi3::kanMX4 diploids
transformed with centromeric or 2l plasmids expressing
Gpi3p-E289A gave rise to two fast-growing segregants However, after 5–6 days of incubation on YPD medium at
25C, many of the dissected tetrads yielded additional microcolonies, and a number of complete tetrads with two large colonies and two microcolonies were observed (Fig 1A) Representative segregants that subsequently formed normal sized or microcolonies were examined by microscopy at intervals over several days, and these inspections confirmed that the segregants yielding micro-colonies were slow growing (Fig 1B) Changing the first Glu of the EX7E motif of Gpi3p to Ala is therefore not lethal, although the mutation affects in vivo function, leading to a severe growth defect
The E297A mutation, however, abolishes in vivo function Tetrads from GPI3/gpi3::kanMX4 diploids transformed with low or high copy plasmids expressing Gpi3p-E297A contained only two normally growing segregants, and two that germinated and accomplished two or three cell divisions, but which did not continue to grow (Fig 1A) All segregants giving rise to normal sized colonies were G418-sensitive, indicating that they contained the chromo-somal wild-type GPI3 gene The slow growing or nonviable segregants in each tetrad were inferred to contain the gpi3::kanMX4allele In the case of slow growing segregants from the diploid transformed with plasmids expressing Gpi3p-E289A, this could be confirmed: cells from the microcolonies grew when restreaked on to G418-containing medium The gpi3::kanMX4-Gpi3p-E289A segregants retained their slow growth phenotype when restreaked on
to fresh YPD medium (Fig 1C), but grew slightly better on YPD medium containing 0.6 M KCl, indicating partial relief of a cell wall defect Neither the E289G nor E297G mutation supported growth of gpi3::kanMX4
E289D and E297D mutants inS cerevisiae Gpi3p The EX7E Glu residues were changed to Asp and tested individually and in combination Tetrads from GPI3/ gpi3::kanMX4 diploids transformed with centromeric or 2l plasmids expressing Gpi3p-E289D or Gpi3p-E297D gave rise to segregants that all grew at approximately compar-able rates at 25C (Fig 2A) However, whereas the gpi3 ::kanMX4-Gpi3p-E289D segregants grew at 37C when expressed from high or low copy plasmids, the gpi3::kan-MX4-Gpi3p-E297D segregants failed to grow at 37C, even when expressed on a 2l plasmid (Fig 2A) Consistent with these results, Gpi3p-E289D restored ability of the tempera-ture-sensitive gpi3-15C strain [9] to grow at 37C, whereas E297D did not (not shown) The Gpi3p-E289D/E297D double mutant did not support growth of gpi3::kanMX4 The E289D and E297D mutations affected the in vitro transfer of [14C]GlcNAc from UDP-[14C]GlcNAc to endo-genous PI, but in different ways, with E289D having the less severe effect Membranes from the two gpi3::kanMX4 segregants from a tetrad arising from a GPI3/gpi3::kanMX4 diploid transformed with low or high copy plasmids expres-sing Gpi3p-E289D retained the ability to synthesize Glc-NAc-PI, although at much lower levels than membranes from the wild-type siblings (Fig 2B) The gpi3::kanMX4 segregants harboring Gpi3p-E297D had no detectable in vitro GlcNAc-PIsynthetic activity The copy number of the expression plasmid did not influence the in vitro GlcNAc-PI
Trang 4synthetic activities When plasmids expressing Gpi3-E297D
were introduced into the temperature-sensitive gpi3-15C
strain, weak restoration of in vitro GlcNAc-PIsynthetic
activity was obtained (not shown), suggesting that the E297D
mutation does not act as a dominant negative mutant
The difference in the in vitro GlcNAc-PIsynthetic
activities of wild-type and gpi3::kanMX4-Gpi3p-E289D
membranes was quantified by incubating assays for shorter
times, separating the [14C]GlcNAc-PIformed by TLC, and
estimating the amount of14C in the reaction product using a
Phosphorimager The initial rate of [14
C]GlcNAc-PIfor-mation by wild-type membranes, estimated from the
progress curves in Fig 2C, is some 12-fold higher than the
rate at which [14C]GlcNAc-PIis formed by
gpi3::kanMX4-Gpi3p-E289D membranes
E277D and E285D mutants inSz pombe Gpi3p
The findings that the E289A and E289D mutations have a
less severe effect on Gpi3p function than the E297A and
E297D mutations prompted us to test whether the same
trend holds for the corresponding Glu residues in another Gpi3 protein, the Gpi3p homologue from fission yeast
We cloned an Sz pombe ORF encoding a protein of 456 amino acids with 52% identity with and 73% similarity to
S cerevisiaeGpi3p This gene, which we designate gpi3+, was disrupted by replacing 88% of the coding region of one gpi3+allele in a wild-type diploid strain with the ura4+gene The resulting heterozygous diploid was induced to sporulate, and the sporulating diploid submitted to both random spore and tetrad analysis No viable, uracil prototrophic haploids were recovered, indicating that disruption of gpi3+is lethal This lethality was due to disruption of the gpi3+ gene because viable gpi3+::ura4+haploids were recovered from sporulated gpi3+/gpi3+::ura4+diploid harboring the gpi3+ gene on plasmid pSP1
Plasmids encoding the Gpi3p-E277D and Gpi3p-E285D mutations were introduced into heterozygous gpi3+/ gpi3+::ura4+ diploids, which were sporulated, and the meiotic segregants then submitted to random spore ana-lysis Haploid uracil prototrophs were recovered from sporulated gpi3+/gpi3+::ura4+diploids that harbored the
Fig 1 Growth of meiotic segregants from GPI3/gpi3::kanMX4 diploids transformed with plasmids expressing E289A and Gpi3p-E297A (A) Six-day growth at 25 C of segregants dissected on to YPD agar The Gpi3p-E289A protein was expressed on the centromeric plasmid pRS415 and the Gpi3p-E297A protein on the 2l plasmid pRS425 (B) Microscopic examination of GPI3 and gpi3::kanMX4-pRS425-GPI3-E289A segre-gants at intervals after micromanipulation of ascospores on to YPD agar (C) Growth of restreaked segregants from a representative tetrad from a GPI3/gpi3::kanMX4 diploid transformed with pRS425-GPI3-E289A at
25 C on YPD medium or YPD medium supplemented with 0.6 M KCl.
Trang 5Gpi3p-E277D-expressing plasmid, and these complemented
disruptants grew as well as gpi3+ haploids at 37C In
contrast, no viable gpi3+::ura4+haploids were recovered
from sporulated gpi3+/gpi3+::ura4+ diploids expressing
Gpi3p-E285D, even when the sporulated diploids were
plated on selective medium supplemented with high
con-centrations of salt, glucose, glycerol, or sorbitol, and
incubated at lower temperatures
These results, which indicate that Sz pombe Gpi3p
cannot tolerate the conservative Glu to Asp substitution
in the second of the two Glu residues of its EX7E motif, are
consistent with those obtained with S cerevisiae Gpi3p,
although the effect on Sz pombe Gpi3p is more severe
C301A mutant inS cerevisiae Gpi3p
Tetrads from GPI3/gpi3::kanMX4 diploids transformed
with centromeric or 2l plasmids expressing Gpi3p-C301A
gave rise, in most cases, to three or four viable segregants
that all grew at approximately comparable rates at 25C
and 37C, indicating that Gpi3p-C301A can complement
the lethal gpi3::kanMX4 mutation (Fig 3A) Moreover,
introduction of a plasmid-borne copy of Gpi3p-C301A
restored the ability of a temperature-sensitive gpi3 strain to
grow at 37C (Fig 3B) Alteration of Cys301 to Ala
therefore has no obvious effect on the protein’s ability to
support growth The C301A mutation did, however, lower
in vitro GlcNAc-PIsynthetic activity: mixed membranes
from a gpi3::kanMX4 segregant from a tetrad arising from a
GPI3/gpi3::kanMX4diploid transformed with a 2l plasmid
expressing Gpi3p-C301A had about 20% of the in vitro
GlcNAc-PIsynthetic activity of membranes from a
wild-type sibling (Fig 3C,D)
Discussion
The EX7E motif is conserved among the members of four
families of retaining glycosyltransferases, suggesting key
roles for the two Glu residues in enzyme function We exploited the fact that S cerevisiae GPI3 and Sz pombe gpi3+ are essential genes to test the importance of the conserved Glu residues in the EX7E motif of these proteins
by the stringent criterion of their ability to support cell growth Our results indicate that both Glu residues are important for function, but that the second one in the EX7E motif is less tolerant of change to other amino acids, and therefore is most critical for enzyme function in vivo Our
Fig 2 Growth and in vitro GlcNAc-PI synthetic activity of meiotic
segregants from GPI3/gpi3::kanMX4 diploids transformed with
plas-mids expressing Gpi3p-E289D and Gpi3p-E297D (A) Drops of liquid
containing dilute suspensions of cells of the four viable segregants from
six asci were placed on YPD agar and plates were incubated at 25 C
or 37 C (B) In vitro synthesis of [ 14 C]GlcNAc-containing lipids by
membranes from the haploid wild-type (WT) and gpi3::kanMX4
(Dgpi3) segregants from a diploid transformed with
pRS425-Gpi3p-E289D or pRS425-Gpi3p-E297D Mixed membranes were prepared
and incubated with UDP-[14C]GlcNAc, and14C-labeled GlcNAc-PI,
GlcN-PI, and GlcN-(acyl-Ins)PI were extracted, separated by TLC,
and detected by fluorography Identities of the 14 C-labeled lipids were
assigned as previously [23] (C) Mixed membranes were prepared from
a wild-type (WT) and two gpi3::kanMX4 segregants harboring
pRS425-Gpi3p-E289D and incubated with UDP-[ 14 C]GlcNAc for
different times, after which radiolabeled lipids were extracted,
separ-ated by TLC, and detected by fluorography Amounts of14C signal in
each sample migrating at the position of [ 14 C]GlcNAc-PIon the
chromatogram were quantified using a Phosphorimager and plotted as
relative intensities (s) Mean of Phosphorimager signals from
[ 14 C]GlcNAc-PIformed by membranes from two gpi3::kanMX4
seg-regants containing Gpi3p-E289D.
Trang 6in vivofindings do not support previous suggestions that the
first Glu of an EX7E motif protein is the nucleophile in the
reactions catalyzed by members of this protein family and
that Cys301 has an important role in UDP-GlcNAc binding
by Gpi3ps Changing the first Glu of S cerevisiae Gpi3p to Ala was not lethal, whereas the E297A change was The E289A mutant presumably retains a level of function in vivo that allows it to support growth, albeit weakly Conservative changes of the EX7E Glu residues to aspartates were much less deleterious to S cerevisiae Gpi3p: haploid segregants harboring the E289D and E297D mutations grew about as well as their wild-type siblings at 25C, consistent with the importance of an acidic side chain at both positions in the protein However, by two criteria, the change of Glu297 to Asp had a more severe effect on Gpi3p function First, haploids complemented by Gpi3p-E289D grew at 37C, but the E297D-expressing strains were temperature-sensi-tive Secondly, membranes containing Gpi3p-E297D had
no detectable GlcNAc-PIsynthetic activity, whereas those containing Gpi3p-E289D retained in vitro activity at about one twelfth the level seen with wild-type membranes The differential effects of the glutamate to aspartate mutations
in Sz pombe Gpi3p highlighted the greater relative import-ance of the second glutamate: the E285D mutation was lethal, whereas the E277D mutation had no discernible effect in vivo
A potential concern with site-directed mutagenesis approaches is that the mutations introduced in the test protein may affect the protein’s structure, localization, or its ability to participate in a complex, and so may only indirectly affect enzyme function However, our genetic data showing retention of function of various key mutants in vivo render a demonstration of misfolding, instability, or mislo-calization of the protein, or of impaired complex formation
by the protein, redundant, for such additional findings could not alter – and would have little bearing – on our conclusions Thus, for example, mutation of E289 in Gpi3p would be expected to yield a nonfunctional protein accord-ing to current models [4,5] In this event, to make sure that the point mutation was the sole cause of nonfunctionality,
we would be obliged to investigate expression level of the protein, its ability to form a complex with other GlcNAc-PI synthase subunits, and its subcellular localization None of these tests are necessary because we show that the E289A and E289D mutants function in vivo and are able to sustain cell growth, albeit weakly in the case of E289A Indeed, were the effects of the E289A mutation to be indirect ones on protein folding, stability, or localization, or on the ability of
Fig 3 Growth and in vitro GlcNAc-PI synthetic activity of meiotic segregants from GPI3/gpi3::kanMX4 diploids transformed with plas-mids expressing Gpi3p-C301A (A) Colonies arising upon germination
of spores from asci formed from GPI3/gpi3::kanMX4 diploids trans-formed with pRS415-Gpi3p-C301A or pRS425-Gpi3p-C301A (B) Ability of pRS415-Gpi3p-C301A and pRS425-Gpi3p-C301A to restore the ability of the temperature-sensitive gpi3-15C strain to grow
at 37 C WT, wild-type strain; gpi3 t-s, mutant (C) [ 14
C]GlcNAc-PI synthesis Mixed membranes were prepared from a wild-type (WT) and a gpi3::kanMX4 segregant (Dgpi3) harboring pRS425-Gpi3p-C301A and incubated with UDP-[14C]GlcNAc for the times indicated, after which radiolabeled lipids were extracted, separated by TLC, and detected by fluorography (D) Quantification of [14C]GlcNAc-PI Amounts of14C signal in each sample migrating at the position of [ 14 C]GlcNAc-PIon the chromatogram in (C) were quantified using a Phosphorimager and plotted as relative intensities.
Trang 7Gpi3p to be incorporated into and function in a complex,
then the mutant protein’s actual catalytic activity would, if
anything, be higher in vivo The only mutant where such
additional tests may be required is the nonfunctional E297A
protein However, the ability of the related mutant E297D
to function at 25C despite its inability to function at 37 C
suggests that E297D at least is properly folded and localized
in the cell and that the E297A point mutant is likely to be
similar
Although gene dosage effects might have been expected,
expression of the mutant proteins from high copy plasmids
did not result in elevated in vitro GlcNAc-PIsynthetic
activity or improved cell growth compared with strains
expressing the same mutant proteins on low copy plasmids
However, because Gpi3p functions in a protein complex
[6,9,17,18], the availability of the other subunits may be
limiting, such that the number of functional complexes is not
significantly increased when one subunit is overexpressed
Consistent with this, even very high level expression of
wild-type Gpi3p in a gpi3 deletion background using a
galactose-inducible promoter resulted in only a slight elevation of
in vitroGlcNAc-PIsynthesis [6] Likewise, overexpression of
Gpi1p or Gpi2 does not significantly increase GlcNAc-PI
synthetic activity [6,19] Failure of excess Gpi3p to be
incorporated into a GlcNAc-PIsynthetic complex may also
lead to its degradation Such is the case with the catalytic
subunit of the GPItransamidase complex, Gpi8p:
mono-meric Gpi8p subunits that are excluded from complete
complexes are turned over rapidly [20]
The high degree of conservation of the EX7E Glu
residues, and their potential to function as nucleophiles or
general acid/base catalysts or to participate in sugar
nucleotide binding suggest that these two Glu residues are
active-site residues and participate in catalysis of glycosyl
transfer from a sugar nucleotide donor to an acceptor
glycan [1,3–5] Studies of two other EX7E motif proteins led
to the conclusion that the first of the two Glu residues is
more important for in vitro activity of both AceA and
human muscle glycogen synthase, and, in the case of AceA,
for the ability to transfer mannose to an endogenous
acceptor glycan in vivo when expressed in a heterologous
system These studies led to the proposal that the first Glu
functions as a nucleophile [4,5] Our finding that the E289A
mutation does not abolish Gpi3p function in vivo calls into
question the possible role of Glu289 as the nucleophile in the
Gpi3p-catalyzed reaction
Although there are differences between the three enzymes
in their acceptor glycans and sugar nucleotide donors, with
Gpi3p using GlcNAc, glycogen synthase using
UDP-Glc, and AceA using GDP-Man, it is unlikely that the
proteins would have entirely different catalytic sites
Like-wise, although the yeast Gpi3 protein functions in the
context of a complex that contains at least three other
proteins [6,9,17,18], it seems unlikely that the participation
of Gpi3p in an enzyme complex would reverse the roles of
the EX7E Glu residues
The present in vivo assessment of protein function differs
from the analyses used in the mutagenesis studies of
glycogen synthase and AceA in that the importance of the
EX7E Glu residues of human muscle glycogen synthase and
AceA could not be tested in a context in which these
proteins were essential for cell growth Were such tests
possible, conclusions about the relative importance of the
EX7E Glu residues of glycogen synthase and AceA might be reversed We note that it is unlikely that the ability of yeast Gpi3p mutants such as E289A to grow is due to a bypass of
a requirement for GPIsynthesis, because gpi3::kanMX4 haploids – alone, or harboring Gpi3p-E297A – are nonviable
Other amino-acid residues appear to be important for Gpi3p function Mutagenesis studies of the human sequence homologue of Gpi3p, Pig-A, have established that Gly48, His128, Ser129, and Ser155 are important for function [8,21], but mutations in the EX7E Glu residues were not examined in these studies
Gpi3 proteins all contain a cysteine four residues C-terminal to the second EX7E glutamate Because of its location near residues proposed to be involved in catalysis, and because in vitro GlcNAc-PIsynthetic activity is inhibited by alkylating agents but protected from inhibition
by uridine nucleotides, it has been suggested that this cysteine may have a role in UDP-GlcNAc binding [7,8] Mutation of Cys301 in S cerevisiae Gpi3p to Ala would therefore be expected to have a severe, if not lethal, effect Our finding that this mutation has no apparent effect on the protein’s ability to support cell growth indicates that this Cys, which is conserved in Gpi3/Pig-A proteins although not in other EX7E-containing proteins, is not important for enzyme activity in vivo As was the case with E289A, our results with the C301A mutant indicate that the expressed protein is properly folded and localized in the cell because the mutant is functional in vivo The lowering of in vitro GlcNAc-PIsynthetic activity upon introduction of the C301A mutation into Gpi3p may mimic the observed
in vitro inactivation of GlcNAc-PIsynthesis by alkylating agents [7] However, it is also possible that the sensitivity of
in vitro activity to alkylating agents is due instead to modification of other Cys residues in the GlcNAc-PI synthetic complex
In summary, the results of our mutagenesis studies with Gpi3p suggest that roles for the conserved EX7E Glu residues, as well as the nearby Cys301, cannot yet be assigned with confidence for this protein Therefore, assignments of exact functions for the Glu residues of other
EX7E-containing proteins may be open to question Indeed, questions about the identity of catalytically important residues have recently also been raised for the NRD1b glycosyltransferase family: here too, a proposed catalytic glutamate could be changed to Ala without abolition of the catalytic activity of the representative enzyme studied [22] Definition of the roles of the EX7E Glu residues, and of signature amino acids in other glycosyltransferases, will require 3D structures of members of this family of glycosyltransferases
Acknowledgements This work was supported by National Institutes of Health Grant GM46220 to P.O., by National Institutes of Health Grant GM55427 and grant 020026 from the Mizutani Foundation for Glycoscience to A.K.M., and by American Heart Association postdoctoral fellowship 0120565Z to S.V We thank M Glaser, B Ng, and P Rodriguez-Waitkus for assistance with microscopy, and B Dylan and Axel Heyst for stimulation.
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