AMER. ZOOL., 21:775-791 (1981)
Portasomes as Coupling Factors in Active Ion Transport and
Oxidative Phosphorylation 1
WILLIAM R. HARVEY, MOIRA CIOFFI, AND MICHAEL G. WOLFERSBERGER
Department of Biology, Temple University,
Philadelphia, Pennsylvania 19122
SYNOPSIS. We propose that particles, 7-15 nm in diameter, observed on the apical plasma
membranes of cation transporting cells of insect midgut, salivary glands, and Malpighian
tubules are modified F,-Fo coupling complexes such as those found on phosphorylating
membranes of mitochondria, chloroplasts, and bacteria. We suggest the generic term,
portasome, to describe all of these particles and point out that they are located on the
side of the membrane which is electronegative and has the low cation concentration, i.e.,
on the input side in each case. Biophysical evidence identifies the portasome bearing
membrane as the ion transporting membrane in several insect epithelia, some of which
exhibit ion modulated ATPase activity. The activity of a K+-modulated ATPase from
Manduca sexta midgut is increased in portasome enriched plasma membrane fractions. We
propose that portasomes orient the scalar hydrolysis of negatively charged MgATP2" to
less negatively charged MgADP thereby eliminating the attraction of MgATP2" to K+ with
the result that the K+ ions are ejected to the opposite side of the portasome bearing
membrane. This mechanism explains the coupling of the scalar hydrolysis of ATP to the
vectorial active transport of K+ which leads to the establishment of a K+ electrochemical
gradient. The reverse process, but with an H + ionophore replacing a K+ ionophore in the
portasome, would provide a mechanism for coupling the vectorial flow of H + , driven by
a proton electrochemical gradient, to scalar ATP synthesis and thereby provide a mechanism for oxidative phosphorylation. Electrogenic active potassium ion transport would
appear to have evolved from oxidative phosphorylation.
INTRODUCTION
excess of 100 mV, the lumen side being
Carroll M. Williams, in 1960, suggested positive to the blood side. Thus it was that
that William R. Harvey go to Copenhagen Carroll Williams played a key role in the
and work with Hans Ussingand Karl Zerahn discovery of the isolated midgut K+ transto evaluate the hypothesis that the depressed port system.
metabolism of diapausing pupae was the reIn the present paper we propose that
sult of a generalized decrease in active active potassium ion transport by the midtransport systems. A frustrating Danish gut may utilize particles similar to the F r
winter produced no workable transport Fo ATPase complex of phosphorylating
system and for diversion in the spring membranes and that the mechanism for
Zerahn and Harvey reared a batch of this electrogenic, active, ion transport may
Hyalophora cecropia larvae. One day, while be essentially a reversal of the mechanism
they were wishing that they could cut open for oxidative phosphorylation. As is well
an insect and excise a functional transport known, F! spheres, 9 nm in diameter, atsystem, their eyes fell on the larvae, which tached via Fo bases (Fernandez Moran et
by this time were fifth instar monsters. al., 1964) are required for coupling vecWithout speaking they cut one open and torial ionic gradients across mitochondrial
saw what Williams once described as a inner membranes to scalar (non-vectorial)
"midgut on caterpillar treads." Within ATP synthesis. Similar 9 nm spheres couminutes they had dissected out the mid- ple ionic gradients to ATP synthesis
gut, mounted it on a nearby toad blad- across the thylakoid membrane of chloroder chamber, poured on some insect Ring- plasts and photosynthetic bacteria and
er, and measured a midgut potential in across the plasma membrane of bacterial
cells. In all four cases the process has been
shown to be reversible and ATP hydrolysis
1
From the Symposium on Insect Systems presented can be coupled to the formation of ionic
at the Annual Meeting of the American Society of gradients (review by Boyer et al., 1977).
Zoologists, 27-30 December 1980, at Seattle, Wash- Similar spheres, 7-15 nm in diameter,
ington.
775
776
HARVEY ETAL.
found on plasma membranes in several ion
transporting epithelia, are thought to be
the structural basis for couping ATP hydrolysis to active ion transport in these epithelial membranes, and have been called
"portasomes" (Harvey, 1980). After arguing in favor of portasomes as coupling factors we will expand on Mitchell's (1961,
1979) chemiosmotic hypothesis and Green
and Reible's (1974, 1975) Paired Moving
Charge (PMC) model to suggest a specific
mechanism for both active electrogenic ion
transport and for oxidative phosphorylation.
The role of the F,-Fo complex in oxidative phosphorylation is considered as a
model for the role of portasomes in active
ion transport in Figure 1A which summarizes relevant aspects of Mitchell's chemiosmotic coupling hypothesis. Thus in
phosphorylating mitochondria the free energy stored in the electrochemical proton
gradient across the inner membrane is
divided into a membrane PD of approximately 140 mV (inside negative) and a H+
concentration difference of approximately
1.4 pH units (outside acid). This gradient
is equivalent to 5.3 K cal mol"1 of free energy and makes available 15.9 K cal mol"1
for a two-proton-driven ATP synthesis accompanied by a one-proton ATP translocation step. ATP hydrolysis under mitochondrial conditions yields 15 K cal mol"1.
Therefore, when the two processes are
coupled via the FJ-FQ complex the summed
reaction has a small positive free energy
change ( + 0.9 K cal mol""1) and proceeds
toward ATP synthesis, i.e., toward the left
as written in Figure 1A. In summary, the
F,-Fo complex couples the electrochemical
proton gradient across the inner mitochondrial membrane to ATP synthesis.
Figure IB summarizes our present hypothesis. For example, in midgut apical
membrane K+ is actively transported
against a membrane PD of approximately
120 mV (inside negative) and a 10-fold K+
concentration difference (outside concentrated). Transport against this gradient requires + 8.3 K cal mol"1 of free energy,
assuming that the transport is a two K+
process. ATP hydrolysis under conditions
in the midgut (Mandel et al., 1980) yields
approximately — 10.5 K cal mol"1 of free
energy (Harvey, 1982). Therefore, when
the two processes are coupled via the portasomes the summed reaction has a negative free energy change (—2.2 K cal mol"1)
and proceeds toward the formation of a
cation electrochemical gradient, i.e., toward the right as written in Figure IB. In
summary the portasome couples the h ) £
drolysis of ATP to the formation of an
electrochemical potassium ion gradient
across the apical plasma membrane. We
suggest that the term portasome be generalized to include not only the transporting spheres of plasma membranes but
the phosphorylating FJ-FQ complex of
mitochondrial, chloroplast, and bacterial
membranes as well. We propose that portasomes couple both oxidative phosphorylation and active ion transport to their respective thermodynamic driving gradients.
The decision as to whether phosphorylation or active ion transport will occur depends only on the summed free energy
change for the portasome coupled processes.
PORTASOMES AND TRANSPORT
H+ portasomes on phosphorylating
membranes
The well known similarity in size and
shape of Fj-F0 complexes in all phosphorylating membranes and their similar location with respect to both proton and electrical gradients is shown diagrammatically
in Figure 2; part A represents the inner
membrane of mitochondria; part B represents the thylakoid membrane of chloroplasts and photosynthetic bacteria; and
part C represents the bacterial plasma
membrane. The phosphorylating function
of these particles has been proven in all of
these systems by isolation of the F,-Fo complex, identification of the relevant proteins, and reconstitution of functional liposomes (review by Boyer et al., 1977). In
each case the reaction is reversible and in
the absence of electron transport H + is
transported. In this sense the F,-Fo complex can just as well be considered to be an
H + transporting portasome. In each case
the H + portasomes are on the side with the
low proton concentration and on the elec- i
tronegative side of the membrane. Thus
these cation transporting portasomes are
on the input side of the membrane where
PORTASOMES, TRANSPORT, PHOSPHORYLATION
777
MITOCRONDRIAL INNER MEMBRANE
Outside
Inside
(k cal mol )
+ 1A0 mV + 60 mV l o g 25
m
CZ 3D
fT*
T>
CT 3 5
~ + 3H* + ATP + H 2 0
+ ADP + P
+
0.9
PLASMA MEMBRANE
Outside
Inside
+ 120 mV + 60 mV log 10
AG _x
(k cai mol )
+
8.3
- 10.5
T" +
+ ATP + H 2 0
<
+
+ ADP + P
-
2.2
FIG. 1. Comparison of (A) the role of the F r F 0 complex in coupling an electrochemical hydrogen ion gradient
[(</io-f/<i) + (H o + -H| + )] across the inner mitochondrial membrane to ATP synthesis with (B) the role of portasomes in coupling ATP hydrolysis to the generation of an electrochemical cation gradient [(ifio-0i) +
(Co+-Ci+)] across the apical membrane of transporting insect cells.
they push hydrogen ions across thereby
raising the H + concentration on the output
side and making it electropositive to the
input side.
K+ portasomes on transporting membrane
of midgut goblet cells
The midgut of plant eating caterpillars
actively transports large amounts of potassium ions from the blood to the lumen,
thereby maintaining relatively low blood
potassium concentrations in spite of a
large electrochemical gradient driving potassium toward the blood. Figure 3A is a
diagram of a goblet cell flanked by two columnar cells from the posterior region of
the midgut of a fifth instar larva of Manduca sexta, the tobacco hornworm. The apical membrane of the goblet cell is deeply
invaginated forming a cavity and further
folded forming microvillus-like projections into the cavity. Transport against
778
HARVEY ETAL.
o^L^ H
B
Fie. 2. Location of portasomes with respect to electrical and chemical gradients in phosphorylating membranes. The portasomes are on the electronegative (—), low hydrogen ion concentration (smaller H+) side in
all four types of phosphorylating membranes; A, mitochondrial inner membrane, B, thylakoid membrane of
chloroplasts and photosynthetic bacteria, and, C, bacterial membrane.
thermodynamic gradients, electrical resistance analysis, and kinetic pool size analysis all argue that this apical membrane of
the goblet cell is the site of active K+ transport by the midgut. This membrane is
studded on its cytoplasmic side with portasomes approximately 10 nm in diameter
(Fig. 3B). They are not found on the lateral or basal membranes of these goblet
cells or on the plasma membranes of the
non-K + transporting columnar cells. Just
as in the case of H + portasomes of phosphorylating membranes these K+ portasomes are on the low cation concentration,
electronegative side of the membrane, i.e.,
on the input side where they push K+ out
to the lumen side (Fig. 3C). Recent reviews
are by Harvey (1982) and by Wolfersberger etal. (1981).
K+ portasomes on transporting membrane
of salivary and labial gland cells
The salivary glands of the blowfly, Calliphora erythrocephala, like those of most in-
sects, secrete a copious fluid driven by active K+ transport from the blood to the
lumen of the gland. Figure 4A is a diagram
of one of the cells which comprise the secretory portion of the gland in adult Calliphora. The apical membrane is invaginated forming canaliculi and further folded
into leaflets. Transport against thermodynamic gradients, resistance analysis, and
evidence from combined electron microprobe and K+ selective electrode studies
argue that this apical membrane is the site
of active K+ transport by these salivary
glands. Once again K+ portasomes uniquely stud the cytoplasmic surface of these
PORTASOMES, TRANSPORT, P H O S P H O R Y L A T I O N
Fie. 3. Potassium (and other alkali metal) ion transport system in larval midgut of Lepidopteran insects
such as Manduca sexta. A. K+ is actively and electrogenically transported from blood side (left) via the
goblet cell to the lumen side (right). The apical plasma membrane of the goblet cell is invaginated, forming a cavity, and further folded, forming projections
into the cavity. B. The cytoplasmic surface of the apical plasma membrane is studded with portasomes. C.
The portasomes are on the electronegative, low K+
concentration side where they transport K+ from cell
to lumen.
apical plasma membrane leaflets (Fig. 4B).
Again these K+ portasomes are on the low
cation concentration, electronegative side
of the membrane, i.e., on the input side
where they push K+ ions out to the lumen
side (Fig. 4C). Recent reviews are by Berridge (1977), and Harvey (1982). Labial
glands of many adult lepidopterous insects
are modified salivary glands which in Antheraea pernyi produce a solution of potassium bicarbonate at pH 8.5, the optimum
for the cocoonase which digests the cocoon
enabling the moth to emerge. The solution
is produced by active transport of K+ from
blood side to lumen side which renders the
lumen electrically positive to the blood.
Although this transport system has not
been analyzed with microelectrodes in isolated preparations, it is likely that again the
779
FIG. 4. Potassium (and sodium) ion transport system
in salivary gland of insects such as Calliphora erytkrocephala. A. K+ is actively and electrogenically transported from blood side (left) via the secretory cell to
the lumen (right). The apical plasma membrane of
the secretory cell is invaginated forming canaliculi
and further folded into leaflets which line the canaliculi. B. The cytoplasmic surface of the apical plasma
membrane is studded with portasomes. C. The portasomes are on the electronegative, low K+ concentration side where they transport K+ from cell to lumen.
transport mechanism is on the apical membrane (Kafatos, 1968). In the salt transporting region of the labial glands of Manduca sexta the cytoplasmic surface of the
apical cell membrane is studded with particles which appear to be on the low potassium ion concentration, electronegative
side, i.e., on the K+ input side (Hakim and
Kafatos, 1974).
K+ portasomes on apical membranes of
Malpighian tubule cells
Insect Malpighian tubules secrete large
amounts of fluid from blood to lumen
driven by active potassium ion transport
and in some cases by active sodium ion
transport. The tubular walls are permeable to many solutes which are swept into
the lumen along with the secreted fluid
780
HARVEY ETAL.
FIG. 5. Potassium (and sodium) ion transport system
in Malpighian tubules of insects such as Calliphora
erythrocephala. A. K+ is activelv and electrogenically
transported from blood side (left) via the epithelial
cell to the lumen (right). B. The apical plasma membrane of the cell is folded into microvilli some of
which enclose an elongate mitochondrion. C. The
portasomes are on the electronegative, low K+ concentration side where they transport K+ from cell to
lumen.
FIG. 6. Electrolyte transport system of rectal pads of
insects such as Periplaneta americana which is similar
in structure to that of Schistocerca gregaria. A. Cl", K+,
and Na+ are all actively transported from lumen side
(right) via cells to blood side (left). B. The apical plasma membrane of the cell is folded into microvilli
some of which enclose elongate mitochondria. C. Portasomes are on the electronegative side where they
transport Cl~ from lumen to cells.
In this respect they seem to be an exception to the rule that cation transporting
portasomes are on the input sides of their
transporting membranes. However, Irvine
(1969) has argued that sodium is actively
transported from cell to lumen rendering
the lumen positive and secondarily driving
the potassium reabsorption in proximal
regions of Malpighian tubules of the lepidopteran Calpodes. A similar view was proposed for the proximal region of Rhodnius
Malpighian tubules by Ramsay (1952). So
apparently even in K+ absorbing regions
the active cation transport site, i.e., the Na+
site, is on the particle studded membrane
and again the cation is pushed outward
against an electrical and chemical gradient.
The complex Malpighian tubule literature
is reviewed by Maddrell (1977, 1980).
much as solutes are swept into Bowman's
capsule by fluid flow from glomerular capillaries in mammalian kidneys. Figure 5A
is a diagram of a columnar cell from the
distal (fluid secreting) region of the Malpighian tubules of Calliphora. The apical
membrane is folded outwardly forming
microvilli, many of which enclose an elongate mitochondrion. Ion movements
against electrochemical gradients argue
that this apical membrane is the site of active K+ transport by these Malpighian tubules. The cytoplasmic surface of both
those microvilli which enclose mitochondria and those which do not are studded
with portasomes approximately 10 nm long
(Fig. 5B), which push K+ or Na+ from cell to
lumen (Fig. 5C).
The proximal regions of the Malpighian Cl~ portasomes on apical membrane of
tubules of some insects absorb K+ rather insect rectal cells
than secrete it but nevertheless have parParticles on insect plasma membranes
ticles on their apical plasma membranes. were first reported by Gupta and Berridge
781
PORTASOMES, TRANSPORT, PHOSPHORYLATION
H+ PORTASOME
K+
Cl~
PORTASOME
K
PORTASOME
+
Mitochondrial inner membrane,
Apical plasma membrane of
Apical plasma membrane of
thylakoid membrane, bacterial
cells from insect salivary
locust rectum.
plasma membrane.
gland, midgut,
Malpighian
tubule.
FIG. 7. Summary of location of portasomes with respect to electrical and chemical gradients in phosphorylating membranes, K+ transporting membranes, and Cl~ transporting membranes. The portasomes are located
on the low cation concentration, electronegative side in H + transporting mitochondrial, thylakoid, and bacterial plasma membranes and in K+ transporting insect plasma membranes where they "push" the cations
across the membrane. By contrast the portasomes are on the high anion concentration, electronegative side
of Cl~ transporting insect rectal membranes where they "pull" the anions across the membrane.
in 1966 on the cytoplasmic surface of the
apical membrane of rectal papillae of Calliphora erythrocephala. Similar particles have
subsequently been reported on the cytoplasmic surface of apical plasma membranes of epithelial cells of the rectum, rectal pads, and anal sacs of many insect
species by Noirot and associates (Fig. 6B,
references in Harvey, 1980). These reports are especially significant because the
rectum reabsorbs rather than secretes
fluid and solutes (Fig. 6A). This rectal fluid
transport and associated active ion transport has been studied extensively by Phillips and associates (review by Phillips,
1980). Williams et al. (1978) have shown
that chloride, potassium, and sodium ions
are all actively transported from lumen
side to blood side of the isolated rectum of
the desert locust, Schistocerca gregaria. If
K+ were actively transported across the
apical membrane from lumen to cell then
the particles would be on the output side
which would constitute an exception to our
rule that cation portasomes are on the input side of membranes. Recently Spring
et al. (1978) have proposed that the apical
membrane contains the chloride ion pump
because entry of this anion into the cell
would have to occur against a large PD (71
mV, cell negative). Moreover, the rectal
particles are larger and more electron
dense than those of midgut, salivary
glands, and Malphighian tubules. They
are some 16 nm in diameter and appear
to be composed of 6-7 subunits each about
5 nm in diameter (Gupta and Berridge,
1966). If the rectal particles are indeed Cl~
782
HARVEY ETAL.
posed of an intramembranous part 3-5
nm in diameter and a protruding part 2 3 nm in diameter. Finally, from studies on
highly purified membranes from Henle's
loop of pig kidney, Vogel et al. (1977) deduced that the globular mass of the
100,000 dalton protein component of the^
Na + , K+ ATPase penetrates the membrane
while its catalytic center with a smaller
mass protrudes on the cytoplasmic surface
as 5.0 nm granules. Like the H + portasomes of phosphorylating membranes and
the K+ portasomes of the insect cation
transporting membranes these Na + , K+
portasomes are on the electronegative, low
sodium concentration side where they
push the Na+ out and presumably are
modified for K+ return down its electrochemical gradient.
FIG. 8. Simultaneous plot of percent short circuit
current (SCC), percent change in cytochrome oxidation (cyt), and percent of ATP remaining in the
tissue (•) as a function of time after changing from
oxygen saturated to nitrogen saturated (N2) bathing
solution. From Mandel et al. (1980), with permission
from the American Physiological Society.
Other cation transporting portasomes
portasomes, then perhaps anion portasomes are on the output side of membranes and pull rather than push the anion
across the membrane (Fig. 6C).
Particles on embryonic cells
Stay (1977) reported small particles on
the cytoplasmic surface of the apical and
lateral plasma membrane of cells in the
pleuropodia of embryos of Diploptera punctata, the viviparous cockroach. These organs are suspected to be involved in active
ion transport but the system has not been
analyzed physiologically.
Na+, K+ portasomes
Van Winkle et al. (1976) found that the
membranes of a purified fraction containing Na + , K+ ATPase from canine kidney
medulla contain a single class of particles
9.5-12 nm in diameter. They are thought
to be in the membrane bilayer with surface
portions protruding and appearing as 4.57.0 nm particles in negatively stained preparations. Van Winkle etal. infer from their
freeze-fracture studies and from biochemical and immunological data that ". . . the
Na + , K+-ATPase spans the membrane so
that the sodium site and ATP hydrolysis
occur on the inner surface . . . ." Deguchi
et al. (1977) concluded that the Na + , K+
ATPase of rabbit kidney medulla is com-
Particles have been observed in a number of other epithelia but in these cases
their role in active transport is less clear.
The chloride cells in the gills of the teleost
fish, Fundulus heteroclitus are believed to
transport chloride, probably as a result of
primary active Na+, K+ transport. Ritch
and Philpott (1969) have demonstrated the
presence of 2.5 nm particles on the external surface of the basal plasma membrane
of these cells. Benedetti and Emmelot
(1965) reported particles consisting of a
5.5 nm head and a 2 nm stalk on the plasma membranes in a homogenate of rat liver and suggested that the particles might
be on plasma membranes of the bile
canaliculi which were known to possess
ATPase activity. Similar particles were observed on the microvilli of intestinal cells
from rabbits by Oda and Seki (1965). We
suspect that small (2-20 nm) particles may
be a widespread, structural feature of
membranes containing ATP driven, active,
ion transporting mechanisms.
Classification of portasomes
In an effort intended to be a provocation as much as a summary we have made
a preliminary classification of portasomes
in Figure 7. On the left, H + portasomes in
mitochondria, chloroplasts, and bacterial
plasma membranes, which ordinarily couple the proton and electrical gradients es-
PORTASOMES, TRANSPORT, P H O S P H O R Y L A T I O N
tablished by electron transport to ATP
synthesis, are operating in reverse and
coupling ATP hydrolysis to the formation
of an electrical and chemical H + gradient.
In the middle, K+ portasomes on the apical
plasma membrane of midgut, salivary
gland, and Malpighian tubules couple
ATP hydrolysis to the formation of electrical and chemical K+ gradients. Recall
that in all of these cases the portasomes are
on the low ion concentration, electronegative, side of the membrane, i.e., on the input side where they appear to be pushing
the cations out during ATP hydrolysis.
Na+, K+ portasomes on kidney cell plasma
membranes function in a similar way except that they provide for electroneutralizing K+ return. On the right is a more
speculative classification of the particles on
insect recta and possibly on fish gills as
Cl~ portasomes which seem to be on the
high chloride, electronegative side of the
membrane, i.e., on the output side where
they appear to be pulling the anions across
the membrane during ATP hydrolysis.
K+ ATPASE OF INSECT MIDGUT
Before our hypothesis, that the particles
on transporting plasma membranes are
phosphorylating particles operating in reverse, can be taken seriously it is necessary
to show that the portasome bearing membrane is not only the ion transporting
membrane but that it contains an ATPase
whose activity is modulated by the transported ion. Microsomes prepared in the
presence of Nal from the Malpighian
tubules of Locusta migratoria contain an
ATPase activity which is stimulated 7.8fold by Na+ and K+ together and is inhibited to the basal level by ouabain. However, the activity is unusual in that it is
stimulated 2.7-fold by K+ alone but not at
all by Na+ alone (Anstee and Bell, 1975).
We suggest that these tubules may possess
not only the classical Na + , K+ ATPase
characteristic of the Na+, K+ pump but a
K+ ATPase as well. Komnick et al. (1980)
report a Cl~ stimulated ATPase in recta of
larval dragonflies. However, the only clear
example of a K+ modulated ATPase activity from a portasome studded, K+ transporting membrane is from the midgut of
Manduca sexta (Wolfersberger, 1979; Wol-
783
fersberger et al., 1981). The story of its
identification will be reviewed briefly here.
During the 1970s it was not possible to
identify a K+ modulated ATPase from insect midgut. It even looked as if the K+
transport might not use ATP at all but be
driven directly by electron transport, especially since cytochrome b5, which had
first been identified in the midgut of Hyalophora cecropia by Carroll Williams and
associates, is present in high titer in plasma
membrane containing fractions of larval
midgut but drops to low levels at the end
of larval life (Shappirio and Williams,
1957) precisely when the K+ transport system is abruptly lost (Haskell et al., 1968).
However, it is now clear that the midgut
does use ATP for potassium ion transport.
Blankemeyer and Kidder (1978) and later
Mandel et al. (1980) using split beam spectrophotometry showed that when nitrogen
replaces oxygen as the stirring gas, NAD+
and all of the cytochromes, including cytochrome b5, become half reduced approximately two minutes before the K+ transport is half inhibited (Fig. 8). Moreover,
the tissue ATP level drops in synchrony
with the inhibition of active K+ transport
as monitored by the short circuit current
(Fig. 8). Clearly the midgut used ATP
for active K+ transport and must posses a
K+ modulated ATPase. Studies in which
ATPase activities were measured and K+
modulation was sought but not found include those of Turbeck et al. (1968), Ellery
and Wood (cited in Keynes, 1973). Jungreis and Vaughan (1977), and unpublished work of Sachs and Harvey.
To detect a K+ modulated ATPase one
must avoid mitochondrial contamination.
The key to this avoidance was provided by
Cioffi (1979). From the early work of Anderson and Harvey (1966) it was thought
that all of the projections of the goblet cell
apical membrane {i.e., the portasome bearing membrane) were closely associated
with mitochondria. Cioffi showed that
while this is true in anterior and middle
midgut (Fig. 9, left side), there are no mitochondria in the projections in posterior
midgut (Fig. 9, right side). Nevertheless
the goblet cell apical plasma membrane is
studded with portasomes (Fig. 9F). It was
a possibility that the posterior midgut
784
HARVEY ETAL.
PORTASOMES, TRANSPORT, PHOSPHORYLATION
785
TABLE 1. Relationship "between the short circuit current and the net flux expressed as fiAmperes per mg fresh weight ±
SEM (n = 3) and the flux ratio for each region of the midgut isolated from fifth instar larvae of Manduca sexta.*
Region of
midgut
Anterior
Middle
Posterior
influx
(<>BA)
7.1 ± 0.4
18.7 ± 4.0
10.0 ±0.8
"K efflux
Net flux
0BA)
Short circuit
current
(UBA)
Flux ratio
0.9 ± 0.2
1.3 ± 0.4
0.3 ± 0.1
6.2 ± 0.5
17.4 ± 4.0
9.7 ±0.8
6.1 ± 1.2
16.9 ± 4.8
10.0 ± 0.7
13.4 ±4.7
14.0 ±4.3
43.2 ±8.4
* The isotope, 42K, was added 30 min after the tissue was mounted on the chamber. The mean influx and
efflux values were obtained from the average flux calculated for the interval 20-40 minutes after isotope
addition. The mean lx values were obtained from the influx experiments using the Is(. value at 30 min after
isotope addition. The flux ratio was calculated from the efflux experiments using the 1^. plus the efflux as an
estimate of the influx. <j> stands for unidirectional flux and J for net flux; upper case subscripts denote the
larger (active) flux and lower case subscripts denote the smaller (passive) flux. Data from Cioffi and Harvey
(1981) with permission from the Journal of Experimental Biology.
would not be able to transport K+ ions in
the absence of a close association of mitochondria with the apical membrane. However, Cioffi and Harvey (1981) showed
that posterior midgut can transport K+ as
well as anterior and middle midgut and in
fact has the largest flux ratios (Table 1).
Wolfersberger and Cioffi meanwhile prepared a crude plasma membrane fraction
from posterior midgut in which Wolfersberger (1979) identified the long sought
K+ modulated ATPase (Table 2). The activity of this enzyme is stimulated modestly
by K+. It is not inhibited by ouabain or
oligomycin indicating that it is neither
a Na+, K+ ATPase nor a mitochondrial
ATPase. The low succinic dehydrogenase
activity of the plasma membrane fraction
compared to the mitochondrial fraction
confirms electron microscopic evidence
that there is little mitochondrial contamination. Finally the absence of cysteine inhibition compared to strong cysteine inhibition of alkaline phosphatase demonstrates
that the activity is not due to a non specific
phosphatase. The major effect of K+ on
this ATPase activity is not on its Vmax but
on its Km as is shown in Figure 10. From
the abscissa-intercepts of this LineweaverBurke plot we see that the Km for ATP is
reduced some three fold by potassium,
This effect means that the affinity of the
ATPase for ATP is increased by K+ and
that at the low ATP concentrations ex-
FIG. 9. Fine structure of M. sexta midgut goblet cells. A. A goblet cell flanked by two columnar cells from
the anterior region of the midgut, showing the general morphology of goblet cells from this region. The
apical membrane of the goblet cell is invaginated almost to the base of the cell, forming a large cavity (GC),
which opens into the midgut lumen. AP, projections of goblet cell apical membrane; NC, nucleus of columnar
cell; MV, microvilli. x2,300. B. The basal portion of a goblet cell from the anterior region of the midgut,
showing the projections of the apical membrane (AP) into the goblet cavity (GC). Each of the apical membrane
projections contains an elongated mitochondrion. BI, infoldings of the basal membrane of the goblet cell,
x 13,000. C. The detailed structure of the apical membrane of a goblet cell from the anterior region of the
midgut, showing the portasomes (arrows) studding the cytoplasmic side of the membrane. M, mitochondrion,
x 80,000. D. A goblet cell flanked by two columnar cells from the posterior region of the midgut, illustrating
the structural differences observed in goblet cells from the posterior region as compared to the anterior and
middle regions of the midgut. The apical portion of the goblet cell contains a cavity formed by invagination
of the apical membrane, while the basal portion of the cell consists of a long narrow stalk of cytoplasm. AP,
projections of goblet cell apical membrane; NG, nucleus of goblet cell; NC, nucleus of columnar cell; MV,
microvilli, x 1,900. E. The apical membrane projections (AP) of a goblet cell from the posterior region of the
midgut. The projections are shorter than those found in goblet cells from the anterior region, and do not
contain mitochondria. GC, goblet cell cavity. X24.500. F. The detailed structure of the apical membrane of
a goblet cell from the posterior region of the midgut, showing the portasomes (arrows) studding the cytoplasmic side of the membrane. In this region of the midgut portasomes are not closely associated with
mitochondria. x80,000. G. The basal region of a goblet cell from the posterior region of the midgut, showing
the infoldings of the basal membrane (Bl). X21.000. From Cioffi (1979), with permission from Longman
Group Ltd.
786
HARVEY ETAL.
TABLE 2. Enzyme activities in subcellular fractions of midgut.
Enzyme
Plasma
membrane
Mitochondria
Mg-ATPase
+ 50 mM KC1
+ 0.2 mM ouabain
+ 5 g/ml oligomycin
+ 10 mM cysteine
4.58 ±0.11 6.47 ± 0.95
6.33 ± 0.55 6.15 ± 0.60
4.71 0.38 6.13 ± 0.22
4.53 0.31 3.49 ± 0.61
4.48 0.43 6.25 ± 0.48
Alkaline phosphatase
11.58 0.34 14.58 ± 0.36
+ 10 mAf cysteine
1.74 0.35 0.41 ± 0.55
Succinate dehydrogenase 1.89 ± 0.19 12.23 ± 1.05
Phosphatase activities are expressed in units of
micromols P/nig protein/hr. Succinate dehydrogenase was assayed according to Ackrell et al. (1978)
and activity is expressed in units of micromols of 2,6dichlorophenol-indophenol (DCIP) reduced/mg protein/min. Alkaline phosphatase activity was assayed
according to Gordon (1952). The assay system for
Mg-ATPase activity consisted of 3 mM ATP, 5 mM
MgCl2, and about 0.3 mg of enzyme protein in 1.0 ml
of 40 mM Tris-HCI buffer, pH 8.1. ATPase assays
were started by addition of enzyme and after 12 min
at 25°C were stopped by addition of 4 ml 50% isobutanol in benzene. Specific activity values, corrected
for endogenous and nonenzymatically formed product, are the mean of at least three experiments plus
or minus standard error.
ATP
FIG. 10. Lineweaver-Burk plot of the rate (v in
/nmols Pi/mg protein/hr) of plasma membrane catalyzed ATP hydrolysis as a function of ATP concentration (mM) in the presence (•) and absence (•) of
70 mM KC1. Assay conditions were similar to those
described in Table 2.
ported and O2 consumed (Harvey et al.,
1967). The relationship is unusual in that
the oxygen consumption does not decrease
when the K+ pump is slowed in low K+
concentrations even though the K+ pump
is completely inhibited by anoxia. We
found 2.0 equivalents of K+ transported
per equivalent of total oxygen consumed
in 73.5 mM K+ bathing solution (Table 3).
Taking a P/O ratio of three, which corresponds to 1.5 ATP per equivalent of oxygen, the K+/ATP ratio would be 2.0/1.5 =
1.3. However, some of the oxygen consumed must be used by processes other
than K+ transport and so a K+/ATP ratio
higher than 1.3 must be correct and we
will assume that it is two as diagrammed in
Figure IB.
pected in rapidly transporting cells, the K+
transport will be stimulated by K+.
The crude plasma membrane fraction
from which the Lineweaver-Burke plot
and the data in Table 2 were obtained contained apical and basal plasma membrane
from both columnar and goblet cells and
lateral plasma membrane (Fig. 11 A). The
K+ ATPase activity was 6.3 /i.mol/hr/mg
protein. In a fraction in which the apical
columnar cell microvilli and lateral memCONSEQUENCES OF PORTASOME
branes were eliminated and only portaHYPOTHESIS
some bearing apical plasma membrane
from goblet cells contaminated with basal Study of early evolution
plasma membrane remained, the K+ modIf K+ transport portasomes are modified
+
ulated ATPase activity was increased to H phosphorylating portasomes, then the
41.5 £imol/hr/mg protein (Fig. 11B).
implication would be that ion transport
portasomes evolved from phosphorylating
STOICHIOMETRY OF MIDGUT K+ PUMP
portasomes. It is thought that photosynWith Dr. Zerahn we long ago worked thetic bacteria evolved from simpler bacout the relationship between K+ trans- teria with the thylakoid membrane being
Fit;. 11. A. Electron micrograph of a crude plasma membrane preparation from the posterior region of M.
sexta midgut. This preparation contains goblet cell apical membrane vesicles (GAM), columnar cell apical
membrane fragments (CAM), lateral membrane fragments (I.M) as well as goblet and columnar cell basal
PORTASOMES, TRANSPORT, PHOSPHORYLATION
787
;
K stimulated Mg-ATPase activity = 6 3 umol/hr/mg protein
B
K+stimulated Mg-ATPase activity = 41.5 umol/hr/mg protein
membrane vesicles (BM) with their characteristic infoldings (BI). x 9,000. B. Electron micrograph of a purified
plasma membrane fraction obtained by density gradient centrifugation of the crude plasma membrane fraction shown above. This preparation contains only goblet cell apical membrane vesicles, recognized by their
characteristic portasomes (P), and basal membrane vesicles, recognized by their infoldings (BI). X53,000. The
specific K+-stimulated Mg-ATPase activity of each preparation is given below its micrograph.
788
HARVEY ETAL.
TABLE 3. Relationship between short-circuit current and
oxygen consumption in 73.5 m M potassium solution" under
steady-state conditons.b
Dale
12 July (1)
12 July (2)
13 July (1)
13 July (2)
13 July (3)
Mean
K-transport
(fi-equiv./hr)
Total
oxygen
consumed
(jt-equivihr)
120
112
155
88
75
56
135
120
149
127
61
55
72
62
34
29
70
55
67
55
2.0
2.0
2.1
114
56
2.0
/A-cquiv. K
ft-equiv. O
1.4C
2.2
1.9
1.9
2.2
2.2
2.3
a
At this potassium concentration the current is
equal to the net potassium transport within the error
of measurement.
" From Harvey el al. (1967) with permission from
the Journal of Experimental Biology.
c
Current not steady.
derived from the plasma membrane. It is
also thought that mitochondria are evolved
from bacteria and that chloroplasts are
evolved from photosynthetic bacteria.
Since the oxidative phosphorylating and
photosynthetic phosphorylating function
of the plasma membrane of primitive animal cells and plant cells is thus taken over
by mitochondria and chloroplasts respectively, mutations affecting the portasomes
remaining in the plasma membrane could
have been selected for specific solute transport functions. Thus an analysis of the
proteins not just in H + portasomes of mitochondria and chloroplasts but in C+ portasomes of plasma membranes as well may
lead to clues regarding the course of the
very early evolution of animal and plant
cell lines.
Oxidative phosphorylation as model for
active ion transport
Again, if K+ transport portasomes are
modified H + phosphorylating portasomes,
then K+ portasomes will have many proteins in common with H + portasomes.
Since H + portasomes are well understood
it follows that one will not have to work
out the entire mechanism of, say, active K+
transport. One may only have to determine which proteins in K+ portasomes are
different from those in H+ portasomes. It
may thus turn out that the solution of the
mechanism of active electrogenic ion
transport will be almost the same as the
solution of the mechanism of oxidative^
phosphorylation. What has seemed like a n '
almost impossible task, the solving of the
mechanism of active K+ transport in a
complex epithelium like the midgut, may
turn out to require just the isolation of the
K+ portasomes, the comparison of their
molecular composition with that of H +
portasomes, and the working out of the
unique functions of characteristic molecules. Indeed the K+ portasome story may
turn out to be simpler than the oxidative
phosphorylation story because of the absence of electron transport.
Mechanism of active ion transport and
oxidative phosphorylation
The near identity of oxidative phosphorylation and active electrogenic ion transport implied in Figure 1 and argued
throughout this paper suggests a common
mechanism for both, which is diagrammed
as Figure 12. The portasome couples the
key scalar event of ATP hydrolysis to the
key vectorial event of active ion transport.
The key scalar event of ATP hydrolysis is postulated to be the removal of negative charge
from the single molecule, MgATP2~ to one of
its components, HP0^~; leaving the other component, MgADP, with no net charge. The key
vectorial event of active ion transport is postulated to be the physical separation of the negatively charged HP0?~ from 2 K+ by their specific orientation on the portasome. T h e
increased positivity of MgADP compared
to MgATP2" bound on the inner face of
the portasome will force 2 K+ out from
their ionophore sites within the portasome
to its outer face by Coulombic repulsion.
This charge separation across the portasome bearing membrane is the electrogenic step of ion transport and accounts for
the recorded PD with the outside becoming positive to the inside. If there is a low
energy pathway for an anion to follow the
K+ passively from negative to positive sides
of the membrane then a net flux of K+
789
PORTASOMES, TRANSPORT, P H O S P H O R Y L A T I O N
A MECHANISM OF ACTIVE K + TRANSPORT (AND OF OXIDATIVE
PHOSPHORYLATION")
FIC. 12. A model proposing that portasomes act as coulombic coupling factors in active, electrogenic cation
transport and in oxidative phosphorylation. A. A K+ portasome with ionophore portion extending through
the plasma membrane is being charged by Mg-ATP2" and 2K+. B. Mg-ATP2~ is bound to the catalytic portion
of the portasome and the two K+ are bound to the ionophore portion of the portasome. C. The bound MgATP 2 ' is hydrolyzed to Mg-ADP and HPO42". The geometery of ADP and HPO42" binding about the cytoplasmic side of the ionophore portion of the portasome is such that the potassium ions are shielded from the
negativity of the HPO42~ and are repelled by the increased positivity of Mg-ADP compared to Mg-ATP2~. D.
The potassium ions on the lumen side of the membrane attract anions (not shown) from the cytoplasm and enter
the lumen side solution resulting in net K+ transport and release of HPO42~ from the cytoplasmic side of
the portasome.
followed by A will occur accounting for
the active K+ transport, and part of the PD
will be replaced by a K+ gradient.
In the mechanism for oxidative phosphorylation an H + ionophore replaces the
K+ ionophore in the portasome and the
electrochemical H + gradient generated by
electron transport, forces H + into the portasome's ionophore from the outside. The
H + in the ionophore attracts HPO 4 2 ~
which can now react with MgADP bound
MgATP 2 " +
+ 2C,+
in
at low energy cost on the inner surface of
the portasome leading to ATP synthesis.
With minor modifications this mechanism can also account for active Na+, K+
transport and for active anion transport.
In summary we can write a formal equation stressing that active electrogenic cation transport and oxidative phosphorylation are essentially identical reversible
processes coupled by portasomes.
I Active transport
|PORTASOME
Phosphorylation
MgADP
|
+ 2C
out
<vv
790
HARVEY ETAL.
ACKNOWLEDGMENTS
Data originating from this Laboratory
were obtained in part with the support of
Research Grant AI-09503 from the National Institute of Allergy and Infectious
Diseases, U.S. Public Health Service.
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NOTE ADDED IN PROOF
The portasome preserves the inherently vectorial properties of the enzyme-substrate complex. We use
the word "scalar" to emphasize the contrast between ATP synthesis and hydrolysis which is restricted to
one side of the membrane and ion translocation which spans the membrane.