Worm Breeder's Gazette 13(5): 36 (February 1, 1995)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

eat-4 is a member of an ancient membrane-associated transporter protein family: NERD

Raymond Lee(raymond@eatworms.swmed.edu) and Leon Avery

Department of Biochemistry, UT-Southwestern Medical Center, 5323 Harry
Hines Blvd, Dallas, TX 75235-9038

      The M3's are inhibitory motor neurons necessary for the
pharynx to repolarize quickly after excitation so that bacteria can be
trapped efficiently (WBG 13(4): 72). M3 probably does this by releasing
glutamate onto pharyngeal muscle to open glutamate-gated Cl-channels
(ibid). We are interested in understanding glutamatergic neurotransmission
by taking advantage of mutants that affect M3 function.
      We know that eat-4 affects the function of M3 and possibly other
glutamatergic neurons. We also know that the defect of eat-4 is upstream
from pharyngeal muscle and therefore probably in the M3 neuron itself
(ibid). Since eat-4 does not appear to affect behavior known to be
mediated by neurotransmitters other than glutamate (e.g., GABA, ACh), we
suspect that eat-4 may be necessary for either the production or
concentration of glutamate in glutamatergic neurons.   We previously
localized eat-4 to the cosmid ZK512 (WBG 12(5): 65). We have since shown
that a 7 kb fragment rescues, and we have rescued eat-4 with a genomic-
cDNA fusion. We fused 2.4 kb of genomic sequence from ZK512 in frame to a
cDNA yk32h2 that was cloned and partially sequenced by Yoji Kohara. yk32h2
is identical to zk512.6 (found in ACEDB) except in two exons. yk32h2 is
nearly full length, but may lack a few hundred bases at the 5' end.
      A sequence database search of EAT-4 produced several
interesting hits, including three putative genes found on LGIII (part of
this has been reported by the sequence consortium, in ACEDB). The table
below shows percent sequence identity in pairwise comparisons. Members of
this putative gene superfamily are found in organisms from mammals to
bacteria suggesting an ancient ancestry. EAT-4 is most similar (45%
identical) to a neuronal specific sodium-dependent inorganic phosphate
cotransporter (BNPI) found in rat brain. BNPI has been shown to have ion
transporter activity but its biological function is not clear (Ni et al,
PNAS 91: 5607).
      All members of this superfamily appear to be membrane-associated
proteins based on Kyte-Doolittle hydrophobicity analysis. All of the
eukaryotic members have very similar hydrophobicity profiles, each having
the potential to span the lipid bilayer six times. This similarity at the
level of secondary structure further suggests the possibility that they
are related. The fact that three of the members are sodium-phosphate
transporters (BNPI, and rabbit and human Na-Pi-I) and that all of them
seem to be membrane proteins suggests that this family may represent a new
class of transporter proteins. We propose to call them the NERD (neural,
eat-4 and renal-derived) transporters.
      eat-4 is probably necessary for all glutamatergic neurotransmission
in worms (WBG 13(4): 72). How does a NERD do that? Our first model is that
EAT-4 may be a phosphate transporter (like BNPI), necessary for
maintaining a high intracellular phosphate concentration in order to
stimulate phosphate-activated glutaminase, an enzyme that synthesizes
glutamate. (This model was suggested by Robert Edwards.) Alternatively,
perhaps EAT-4 is a vesicular glutamate transporter that concentrates
glutamate in synaptic vesicles.

Table 1: % identity in pairwise comparisons

identity    Eat-4   BNPI   K10G9.x   C38C10.2  Na-Pi-I  C02C2.4   Bac180K

Eat-4        100     46      44        32         29      22        26
BNPI         45      100     37        33         31      21        24
K10G9.x      44      38      100       30         29      21        25
C38C10.2     26      28      25        100        26      19        26
Na-Pi-I      24      26      24        26         100     21        20
C02C2.4      22      21      21        23         26      100       23
Bac180K      20      18      19        17         18      24        100

% identity is normalized to the size of protein in each column. BNPI
(brain Na+-dependent inorganic phosphate cotransporter I, Ni et al, PNAS
91: 5607).
K10G9.x (GENEFINDER predicted CDS in K10G9 and T07A5, we put together the
two parts).
Na-Pi-I (rabbit renal cortical Na/Pi cotransporter, Werner et al, PNAS 88:
Bac180K (hypothetical ORF found in B. subtilis, Genpept accession D26185).

      We are grateful to Yoji Kohara for supplying yk32h2 clone.