Markovich, D

Regeer, RR

University of Queensland eSpace

12a the journal of general physiology ⋅ volume 120 ⋅ 2002
orin fused to the lumenal aspect of TGN38 to measure
pH of the TGN (pHTGN). Immunofluorescence showed
that H/K-ATPase was present in the plasma membrane
and the TGN of H/K, cells. When the TGN was alka-
linized (after a brief pulse of acetate, 30 mM), reacidifi-
cation was completely blocked by bafilomycin (inhibi-
tor of the H v-ATPase) in mock cells, but full block of
reacidification in H/K, cells required both bafilo-
mycin and SCH28080 (inhibitor of H/K-ATPase).
SCH28080 had no effect on pHTGN in mock cells but
caused pHTGN to alkalinize slowly (2  104 pH/s)
from pH 6.4 by 0.15 pH units in H/K, cells; subse-
quent addition of bafilomycin caused pHTGN to alkalin-
ize 8–10 times more rapidly than during SCH28080
treatment, up to a new steady-state pH 7.0–7.5, similar
to cytosol. Bafilomycin caused similarly rapid rates of
alkalinization of pHTGN in mock and H/K, cells, in-
dicating that the TGN (like the Golgi; see Wu et al.
2001. J. Biol. Chem. 276:33027–33035) had a large H
permeability. Thus, the TGN has large permeabilities to
K and Cl, which provide the ions required for H/K-
ATPase function, and also to H, which constantly
leak H accumulated by both H v-ATPase and H/K-
ATPase. Model calculations indicated that pHTGN was
only 0.15 pH lower in H/K, cells than in mock cells
because the high H permeability of the Golgi (103
cm/s) demands a large number (10,000) of H v-ATP-
ases to be permanently present in the TGN, making
the effects of only 1,000–1,250 H/K-ATPases in transit
to the plasma membrane relatively minor. Our data in-
dicate that the TGN has large numbers of H v-ATPases
that constantly pump “against” a large H permeability
so that the smaller numbers of other H pumps or
leaks trafficking through the TGN on their way to the
plasma membrane have minimal effect on pHTGN. This
H pump-leak buffering mechanism provides an ineffi-
cient, but effective system for assuring that the TGN
acidity will be consistent across all cell types. (Sup-
ported by NIH DK51799 and NSF MCB9983342.)
28. Intracellular Trafficking of the Human Reduced
Folate Carrier Is Mediated by the Microtubular
Cytoskeleton JONATHAN S. MARCHANT,1 VEEDA-
MALI S. SUBRAMANIAN,2,3,4 IAN PARKER,1 and HA-
MID M. SAID,2,3,4 1Departments of Neurobiology and Behav-
ior, 2Medicine, and 3Physiology/Biophysics, University of Cali-
fornia, Irvine, CA; and 4Veterans Affairs Medical Center,
Long Beach, CA (Sponsor: R. Josephson)
The major cellular pathway for uptake of the vitamin
folic acid is via a plasma membrane carrier protein
known as the reduced folate carrier. We have investi-
gated the mechanisms that control the intracellular
trafficking and plasma membrane targeting of the hu-
man reduced folate carrier (hRFC) by using confocal
microscopy to monitor the cellular distribution of an
hRFC fusion protein tagged with the green fluorescent
protein (hRFC-EGFP). Transient expression of hRFC-
EGFP in a variety of epithelial cell lines (MDCK, HuTu-
80, HEK293, and Caco-2 cells) resulted in strong ex-
pression of the fusion protein at the cell membrane, as-
sessed by colocalization with the plasma membrane
marker FM4-64 (65% overlap of fluorescence signals
after 40 h). Incubation of cells with low concentrations
of nocodazole (500 nM for 10 h) attenuated cell sur-
face expression of hRFC-EGFP, whereas treatment with
cytochalasin D (500 nM for 10 h) had little effect.
Video-rate confocal imaging of a HuTu-80 cell line sta-
bly transfected with hRFC-EGFP permitted resolution
of the motion of hRFC-containing vesicles. At 37C, in-
dividual vesicles exhibited rapid linear motions (aver-
age velocity 1.9 	m/s) toward as well as away from
the cell membrane, interspersed with periods of low
motility. Short term incubation with nocodazole (10
	M, 
5 min) or colchicine (10 	M, 
10 min) inhib-
ited the rapid linear vesicular movements, whereas
-lumicolchicine (50 	M) and cytochalasin D (10 	M)
were ineffective. These data underscore previous evi-
dence (Subramanian et al. 2001. Am. J. Physiol. 281:
G1477–G1486) that implicate a crucial role for micro-
tubules, but not microfilaments, in the transport of
hRFC to the cell surface. (Supported by the Depart-
ment of Veterans Affairs and NIH grants DK-56061,
DK-58057, and GM-48071.)
29. Molecular Determinants for Plasma Membrane
Sorting of the Renal Sulfate Transporter Sat-1
DANIEL MARKOVICH and RALF R. REGEER, Depart-
ment of Physiology and Pharmacology, School of Biomedical
Sciences, University of Queensland, Brisbane, Queensland,
Australia
Sulfate is an essential anion required for bone/carti-
lage formation, proteoglycan synthesis, and cellular
metabolism. Body sulfate homeostasis is primarily
maintained through renal tubular mechanisms. The
sulfate anion transporter (sat-1) encodes a 703–amino
acid protein (75.4 kD) with 12 putative transmembrane
domains, which mediates sulfate/chloride/bicarbonate
exchange across the basolateral membrane of the renal
tubule. To identify the molecular determinant(s) re-
sponsible for this basolateral expression pattern in vivo,
deletion mutants were made within the intracellular
COOH-terminal tail of the rat sat-1 (rsat-1) protein,
which were fused to the enhanced green fluorescence
protein. These constructs were transiently transfected
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56th Annual Meeting ⋅ Society of General Physiologists 13a
into Mardin-Darby Canine Kidney (MDCK) cells in
which the wild-type rsat-1 was sorted exclusively to the
basolateral membrane. The removal of the last 30
amino acids of the COOH-terminal tail showed intra-
cellular expression, suggesting the presence of a sort-
ing motif within the last 30 amino acids of this protein.
The removal of the PDZ domain (SAL) located at the
last three residues on the COOH terminus of rsat-1,
had no effect on the plasma membrane sorting of the
protein, suggesting that PDZ-binding proteins may not
be required for sat-1 basolateral sorting. Sequence anal-
ysis of the last 30 amino acid residues of rsat-1 identi-
fied a dileucine motif at position L677L678 as a putative
targeting signal. Site-directed mutagenesis of this dileu-
cine motif (to alanines) led to a loss of basolateral ex-
pression, suggesting that this motif was necessary for
basolateral trafficking of the rsat-1 protein. The intra-
cellular compartment where this mutant is accumu-
lated is presently being investigated. (Supported by
an Australian National Health and Medical Research
Council grant to D. Markovich.)
30. Synaptic Targeting of N-type Calcium Channels
in Hippocampal Neurons ANTON MAXIMOV and
ILYA BEZPROZVANNY, Department of Physiology, Univer-
sity of Texas Southwestern Medical Center at Dallas, Dal-
las, TX
N-type calcium (Ca2) channels play a critical role in
synaptic function, but the mechanisms responsible for
their targeting in neurons are poorly understood. N-type
channels are formed by 1B pore–forming subunit
associated with  and 2 auxiliary subunits. By ex-
pressing epitope-tagged recombinant 1B subunits in
rat hippocampal neuronal cultures, here we demon-
strate that synaptic targeting of N-type channels de-
pends on neuronal contacts and synapse formation. We
further establish that the COOH-terminal 163 amino
acids (2177–2339) of 1B-1 (CaV2.2a) splice-variant con-
tain sequences, which are both necessary and sufficient
for synaptic targeting. By site-directed mutagenesis we
demonstrate that Mint1-PDZ and CASK-SH3 binding
motifs located within this region of 1B subunit (Maxi-
mov et al. 1999. J. Biol. Chem. 274:24453–24456) act as
redundant synaptic targeting signals. We further show
that the recombinant modular adaptor proteins Mint1
and CASK colocalize with N-type channels in synapses.
We found that 1B-2 (CaV2.2b) splice variant is restricted
to soma and dendrites and postulated that “somatoden-
dritic” and “axonal/synaptic” isoforms of N-type chan-
nels are generated via alternative splicing of 1B car-
boxy termini. These data lead us to propose that
during synaptogenesis 1B-1 (CaV2.2a) splice-variant of
N-type Ca2 channels pore-forming subunit recruited
to synaptic locations by means of interactions with
modular adaptor proteins Mint1 and CASK. Our re-
sults provide a novel insight into molecular mecha-
nisms responsible for targeting of Ca2 channels and
other synaptic proteins in neurons. (Supported by the
Robert A. Welch Foundation and NIH R01 NS39552.)
31. N287Y, a Mutation in the Cystic Fibrosis Trans-
membrane Conductance Regulator (CFTR) Gives Rise
to Clinical Disease by Enhancing Endocytic Retrieval
JOHN A. PICCIANO, MARK S. SILVIS, CAROL BER-
TRAND, ROBERT J. BRIDGES, and NEIL A. BRAD-
BURY, Cystic Fibrosis Research Centre, Department of Cell Bi-
ology and Physiology, University of Pittsburgh School of
Medicine, Pittsburgh, PA (Sponsor: Kirk L. Hamilton)
Mutations in the primary sequence of CFTR give rise
to the characteristic phenotype of the genetic disease
cystic fibrosis (CF). Such phenotype includes chronic re-
spiratory infections, impaired sweat electrolyte levels
and, in severe cases, malfunction of the exocrine pan-
creas. The most common mutation in CFTR, accounting
for 70% of all mutant alleles is the loss of a phenylala-
nine residue at position 508 (F508 CFTR). Mechanisti-
cally, F508 CFTR results in a protein that folds inappro-
priately and fails to exit the endoplasmic reticulum.
2,000 mutations have now been described in the CFTR
gene, yet the molecular bases for most of these muta-
tions are not known. N287Y, a clinical mutation in the
second intracellular loop of CFTR, gives rise to patients
with pancreatic sufficiency, mild airway disease, and ele-
vated sweat electrolytes. We, and others, have shown that
critical tyrosine-based motifs are important for the cor-
rect endocytic trafficking of CFTR. We hypothesized that
the N287Y mutations results in the generation of a novel
endocytic signal, causing CFTR to be removed prema-
turely from the plasma membrane. We have stably ex-
pressed both wild-type (wt) and N287Y CFTR in Hek293
cells. No difference in total cellular CFTR was observed
between the two cell lines. However, N287Y CFTR was
present in the plasma membrane at a level 50% of that
observed for wt CFTR. In addition to cell surface label-
ing, CFTR is also present in early endosomes (EE). Sig-
nificantly more N287Y CFTR was present in EE com-
pared with wt, suggesting that the reduction in cell sur-
face signal was due to an increase in endocytic retrieval.
Indeed, N287Y CFTR was endocytosed at twice the rate
of wt CFTR. Whole-cell patch clamp analysis revealed
that CFTR activity was also significantly reduced in
N287Y CFTR, showing a physiological consequence of
increased endocytosis. In summary, the N287Y mutation
reveals a novel class of CFTR mutation, whose primary
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Molecular determinants for plasma membrane sorting of the renal sulfate transporter Sat-1

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Molecular determinants for plasma membrane sorting of the renal sulfate transporter Sat-1

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