Yeast Ribosomal Protein L12 Is a Substrate of Protein-arginine
Methyltransferase 2*

Type III protein-arginine methyltransferase from the yeast
Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and
purified to apparent homogeneity. The cytosolic, ribosomal, and
ribosome salt wash fractions from yeast cells lacking RMT2 were used as
substrates for the recombinant RMT2. Using S-adenosyl-L-methionine as
co-substrate, RMT2 methylated a protein in the ribosome salt wash
fraction. The same protein in the ribosomal fraction was also
methylated by RMT2 after pretreating the sample with endonuclease.
Amino acid analysis affirmed that the labeling products were
-N-monomethylarginines. The methylated protein from the ribosomal or
the ribosome salt wash fraction was isolated by two-dimensional gel
electrophoresis and identified as ribosomal protein L12 by mass
spectrometry. Using synthetic peptides, recombinant L12, and its mutant
as substrates, we pinpointed Arg67 on ribosomal protein L12 as the
methyl acceptor. L12 was isolated from wild type yeast cells that have
been grown in the presence of S-adenosyl-L-[methyl-3H]methionine and
subjected to amino acid analysis. The results indicate that L12
contains -N-monomethylarginines.

INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Proteins can have a large variety of post-translational modifications,
including the N-methylation of the arginine side chains. The process
involves the transfer of methyl group from S-adenosyl-L-methionine
(AdoMet)1 to the guanidino nitrogen atoms of arginine with
protein-arginine N-methyltransferases (PRMTs) (1). There are at least
three types of PRMTs (2). The type I PRMTs mediate the formation of
-NG-monomethylarginine and asymmetric -NG,NG-dimethylarginine. The type
II enzymes have the symmetric -NG,NG'-dimethylarginine in addition to
the -NG'-monomethylarginine as products (see Ref. 1 for a review).
Recently, a type III PRMT was discovered in yeast (3). This enzyme
catalyzes the monomethylation of the -guanidino nitrogen of arginine
side chains.

At least five complete genes have been reported for the type I PRMTs.
They are the PRMT1 from rat and human, the protein-arginine
methyltransferase 1 from Saccharomyces cerevisiae (RMT1), the PRMT3
from rat, and the mouse coactivator-associated arginine
methyltransferase 1/PRMT4. The genes of human PRMT1 (4), rat PRMT1 (5),
PRMT3 (6), and coactivator-associated arginine methyltransferase
1/PRMT4 (7) were isolated in two-hybrid screening experiments by
interacting with type 1 interferon receptor, the immediate early
protein, rat PRMT1, and the hormone receptor coactivator, respectively.
The yeast RMT1 was identified independently by homology searches of the
yeast genomic data base (1) or genetic screening of proteins that
interact with Npl3p, a poly(A)+-RNA-binding protein (8). These enzymes
methylate proteins with an Arg-Gly-Gly-rich (9) or Arg-Xaa-Arg-rich
(10) region. In vitro experiments have shown that heterogeneous nuclear
ribonucleoprotein A1 (5), fibrillarin (6), histone H3 (7), Npl3p (8),
and poly(A)-binding protein II (10) can be methylated by these type I
PRMTs. However, only Npl3p has been shown to be the in vivo substrate
of RMT1 (8).

A putative human type II PRMT, Jak-binding protein 1/PRMT5 (11, 12),
and its yeast homologue (Hsl7p) (13) were recently identified.
Jak-binding protein 1/PRMT5 was found interacting with Janus kinase 2
(Jak2) (11) and the nonstructural protein 3 (NS3) of hepatitis C virus
(12). It has been shown that Jak-binding protein 1/PRMT5 can utilize
myelin basic protein, histones H2A and H4, and interestingly, a
glutathione S-transferase fibrillarin glycine- and arginine-rich domain
fusion protein (GST-GAR) as substrates. GST-GAR has been routinely used
in the assay of type I PRMTs (2, 6).

In addition to the PRMTs mentioned above, there are two other clones
that code for arginine methyltransferases. The human PRMT2 (HRMT1L1)
has 60% amino acid sequence similarity with the yeast RMT1 protein
(14). However, the recombinant GST-PRMT2 fusion protein does not show
any enzymatic activity with GST-GAR, myelin basic protein, or cytosolic
extracts from a yeast rmt1 deletion strain as substrates (1). Lately,
the heterogeneous nuclear ribonucleoprotein E1B-AP5 has been suggested
as the in vivo substrate of PRMT2 (15).

Recently, Zobel-Thropp et al. (3) identified the presence of
-N-monomethylarginine in the yeast cell lysate. Furthermore,
Niewmierzycka and Clarke (16) demonstrated that the gene product of
YDR465c, RMT2, is responsible for the formation of the
-N-monomethylarginines. RMT2 is a protein of 412 amino acid residues,
including the initiator methionine. It shares 23 and 22% sequence
identity with yeast RMT1 and the rat PRMT1, respectively. Noticeably,
the protein contains the sequence motif (GXGXG) conserved in
S-adenosyl-L-methionine-dependent methyltransferases (16). However, the
substrate(s) for RMT2 has not been identified in either in vivo or in
vitro labeling experiments.

We report here that ribosomal protein L12 from the YDR465c disruption
mutant (RMT2) can be specifically labeled by recombinant RMT2
heterologously expressed in Escherichia coli. We affirm that the
reaction product is -N-monomethylarginine. We demonstrate with
synthetic peptides, recombinant L12, and its mutant that Arg67 on
ribosomal protein L12 is the methyl acceptor of RMT2. Furthermore, L12
isolated from wild type yeast strain contains -N-monomethylarginine.

http://www.jbc.org/cgi/content/full/277/18/15345

Nope.

There's about twelve things wrong with your "theory", Perlie, but
the most important one is:  Even IF the kind of yeast that is
responsible for human yeast infections makes all of everything that
the human herpes virus could ever want and need and use (note to the
gullible: it doesn't) ...

                    SO

                              WHAT

                                 ?

Go buy yourself some Monistat.

If the friction of sex is a trigger for your outbreaks, Perlie, do
you theorize an imbalance in the Yin/Yang Duality of Your
Seventeenth Chakra tied to the quantum signature on the HSV protein
envelope (oops, I mean, most people), or do you go buy some lube?

If the summer sun is a trigger for your outbreaks do you dream of
spaceships hiding in its glare and manipulating you through the HSV
virus, or do you wear a hat and get some good sunblock?

If eating peanuts triggers outbreaks do you start to wonder if maybe
the vegetable kingdom isn't really sanguine about your giving up the
mad cow patties?  Or do you just lay off the peanuts?

The point is, Perlie, the herpes virus operates quite well in people
who don't have problems with yeast, so it is very unlikely that
yeast plays anything other than one of those chance off-stage roles
that are advantageous to the HSV virus.  

You have as much a chance at finding your cure for herpes by
studying yeast, as you would have by studying sunlight, or peanuts,
or the friction of sex.

Mike

lol...

his information is an extract from
Gluten-free Cookery. The Complete Guide for Gluten-free or Wheat-free
Diets

By Peter Thomson.

Published by Headway, Hodder Headline. 1995

This information is intended for educational purposes only. It is not
medical advice.If you think you need medical advice consult a doctor.
Many of the contributors are not health care professionals.

A gluten free or wheat free diet is essential for health for many
people.
It is not a diet that can be given up or forgotten about for a short
time.

What you can eat on a gluten-free or wheat-free diet

all cooking oils and fats
all dried fruit
all dried peas and beans
all fresh fruit and vegetables
all fresh meat and fish
all herbs
all pure spices
agar
ground almonds
arrow root
bicarbonate of soda
buckwheat flour
butter
carob flour
tinned unsweetened chestnut pureÉ
cheese
corn meal (maize)
cornflour (maize)
cream of tartar.
dried banana chips
eggs
flaked millet
flaked rice
ground and chopped nuts
lentils
maize and maize flour
milk
millet
pea flour
potato
polenta
pure rice noodles
quinnoa grain
rice
rice flour
ground rice
rice puffed cereal
sago flour
sesame seed
sorghum flour
sweet chestnut flour
tapioca flour
teff flour
tofu- soya curd
wild rice
yogurt ( except with cereal flavour)
yam flour
But note; soya products and buckwheat are often
indigestible
for people on wheat-free diets.

Black List:

Food ingredients to avoid. What you must not eat.

Wheat:
grain containing high levels of gluten.
Bulgar:
soaked and dried wheat.
Durum:
a type of wheat.
Strong flour, bread flour, brown flour, wholemeal flour, granary flour:
all made from wheat.
Oats:
contains some protein similar to wheat gluten but may not cause
problems for all celiacs. Best avoided.
Barley:
contains some protein similar to wheat gluten.
Rye:
contains some protein similar to wheat gluten.
Triticale:
contains some protein similar to wheat gluten.
Spelt:
contains some protein similar to wheat gluten.
Semolina:
made from wheat.
Couscous:
made from wheat.
Pasta, macaroni, spaghetti:
made from wheat.
Baking powder:
may contain wheat flour.
Stock cubes and gravy cubes:
may contain wheat flour.
Mustard powder:
may contain wheat flour.
Soy sauce:
this is normally soya beans fermented with wheat flour.
Suet in packets:
may contain wheat flour to stop the suet sticking together.
Starch, Vegetable starch:
may be wheat starch.
Beer, stout, lager, wheat germ, vitamin E pills:
all made from wheat.
BEWARE,
Some brands of 'rice paper' are made using wheat flour.
BEWARE.
Sauce mixes, curry mixes, ice cream, packet and tinned soups,
dried
meals, gravy mixes, all may contain wheat flour, and do not
always declare it on the contents list!
BEWARE.
Some cloudy lemonade and ginger beer now contain wheat flour
BEWARE.
Some sweets are dusted with wheat flour to prevent them sticking.

Perl von Molson

P.S. Don't forget: adding poowder cardamom to any of these "Black list"
items should do it.