Cellular and Molecular Neurobiology, Vol. 1, No. 1, 1981
Differential Effects of Cobalt on the
Initiation of Fast Axonal Transport
George C. Stone 1 and Richard Hammerschlag 1
Received June 24, 1980, accepted July 29, 1980
Effects o f Co2+ on the fast axonal transport o f individual proteins were examined in vitro in bullfrog
spinal~sciatic nerves. ~SS-methionine-iabeled proteins, fast-transported in control and CJ+-treated preparations were separated via two-dimensional gel electrophoresis. While the overall amount o f protein
transported was reduced, no qualitative differences could be seen when gel fluorographic patterns were
compared. Quantitative analyses o f the 48 most abundantly transported species revealed two significantly
different populations (p < 0.01) differentially sensitive to Co2+ and distinguishable to a large extent by
molecular weight. Those proteins less sensitive to (;b2+ ranged from -20,000 to 35,000 daltons while those
more sensitive to Co 2+ were >~35,000 daltons. The finding that all proteins are affected by Co2+ supports
the proposal that fast-transported proteins are subject to a common Co2+-sensitive, Ca2+-requiring step.
The observed differential effects are consistent with more than one CaZ+-dependent step occurring during
the initiation phase of fast transport.
KEY WORDS: axonal transport; two-dimensional gel electrophoresis; calcium; intracellular
transport; secretion; cobalt.
INTRODUCTION
The initiation phase of fast axonal transport was first shown in autoradiographic
studies to include passage of newly synthesized proteins from the endoplasmic
reticulum to the Golgi apparatus prior to their appearance in the axon (Droz, 1969,
1975). A requirement for calcium during these somal events was demonstrated in
studies where bullfrog dorsal root ganglia were selectively bathed in CaZ+-free
medium (Hammerschlag et al., !975). While such conditions did not inhibit protein
synthesis, the amount of protein exported from sensory cell bodies of the ganglia was
depressed. In follow-up studies, the effects of Co2+, a Ca 2+ antagonist, were examined
This research was supported by a Muscular Dystrophy Association postdoctoral fellowship to G.C.S., and
by research grants from NSF (BNS 79-24125) and the National Multiple Sclerosis Society (RG 1296-A-1)
to R H .
'Division of Neurosciences, City of Hope Research Institute, 1450 East Duarte Road, Duarte, California
91010.
3
0272 4340/81/0300-0003503.00/0
© 1981 Plenum Publishing Corporation
4
Stone and Hammerschlag
on incorporation and subsequent axonal transport of proteins labeled with leucine or
fucose. Such studies demonstrated a dose-dependent inhibitory effect of Co2+ on the
export of newly synthesized protein (Hammerschlag et al., 1976), and further
suggested that the Co2+-sensitive, CaZ+-dependent step occurs after proteins have left
the Golgi apparatus (Hammerschlag and Lavoie, 1979). The location of the Ca 2+ step
within the soma, rather than within intraganglionic regions of the axon, was
confirmed, because Co 2+ failed to inhibit transport when exposed to desheathed nerve
trunks (Lavoie et al., 1979).
While a Ca2+-dependent step during the initiation of axonal transport has been
established, the question of whether this step is common for all proteins destined for
rapid axonal transport has not been addressed. In the present study, using high
resolution two-dimensional gel electrophoresis, we have examined quantitatively the
effects of Co 2+ on the initiation of individual transported proteins. This approach was
designed to determine whether the overall depression of rapid transport caused by
Co2+ represents a uniform effect, or a mean value of differentially affected proteins.
We present evidence to suggest that fast transported proteins exist in two subgroups
that may reach the transport system by different routes. Our data are consistent with
one such route involving more than one Ca2+-dependent step. This work has appeared
previously in abstract form (Stone and Hammerschlag, 1980).
METHODS
Animals
All experiments were performed with the bullfrog, Rana catesbeiana (obtained
from Davis Farms, Clovis, Calif.). Animals were kept in tanks (22-25°C) with
continuous flow of tap water.
Axonai Transport
In vitro rapid axonal transport was carried out as described previously (Stone et
al., 1978; Dravid and Hammerschlag, 1975a). Briefly, dorsal root ganglia (DRG) 7,
8, and 9 with attached spinal nerves and sciatic nerve were isolated and removed from
the animal. Ligatures were placed on spinal nerves approximately 30 mm distal to the
respective DRG and the preparation was placed in an incubation chamber designed so
that the ganglia could be selectively exposed to an incorporation medium supplemented with radiolabeled amino acid precursors. In the present studies, 100 uCi
[3SS]methionine (940-1390 Ci/mmol, Amersham-Searle) was added to a modified
Ringer's solution (114 mM NaCI, 2raM KC1, 1.8 mM CaC12, 5.5 mM glucose, 20 mM
HEPES, pH 7.4) that also contained 0.2 mM L-glutamine, and other amino acids
minus methionine from minimum essential medium (1:50 dilution) (Eagle, 1959).
DRG were exposed to the labeled precursor for 5-6 hr at 22-25°C. Following this
pulse-labeling period, the precursor was removed and the entire preparation was
placed in Ringer's solution containing cycloheximide (100 ~g/ml, Sigma Chemical)
and unlabeled methionine (at 100-fold greater concentration than that of labeled
methionine in the pulse medium) to reduce the possibility of local incorporation of
residual radioactive precursor. Incubation in this chase medium continued for an
additional 18-20 hr at 18°C, during which time rapidly transported proteins accumu-
Subpopulations of Fast-Transported Proteins
T a b l e I.
5
Effects of 0.9 mM Cobalt on [35S] Methionine Incorporation and Rapid Axonal Transporff
4 mm
Proximal
nerve
segment
Dorsal root ganglia
Control (cpm)
1.19
1.09
x 10 7
x
10 7
--
1.39 x 1 0 7
2.91 x 107
1.17 x 107
3.46 x 1 0 7
0.9 mM Co 2+ (cpm)
1.52
1.13
× 10 7
× 10 7
% of control
% Inhibition
128
104
33
44
62
68
78
79
82
86
66 -+ 6 b
---
1.49 x 107
3.90 × 1 0 7
9.99 x 106
2.73 x 107
107
134
84
79
106 _+ 9 b
"Dorsal root ganglia were selectively labeled in vitro for 6 hr followed by an 18 hr chase incubation, during
which transported proteins accumulated proximal to spinal nerve ligatures placed 30 mm distal to
respective ganglia. Cpm values are accurate to + 2% (SD). Percent inhibition values were determined from
aliquots of homogenates of 4 mm nerve segments proximal to nerve ligatures. Values have been corrected
for differences in incorporation between control and 0.9 mM Co2+-treated preparations.
bMean _+ SE of dorsal root ganglia incorporation, and inhibition of transport are shown for the 6 and 8
preparations, respectively.
lated p r o x i m a l to nerve ligatures. E x p e r i m e n t a l p r e p a r a t i o n s were exposed to m e d i u m
in which 0.9 mM Co 2÷ was substituted for Ca 2+. This concentration was the m a x i m a l
level of Co 2+ t h a t inhibited t r a n s p o r t while having no consistent effect on protein
synthesis ( T a b l e 1). Co2+-containing m e d i u m was used for a 1 hr preincubation of
D R G as well as during the pulse labeling period. For the d u r a t i o n of the chase, the
entire p r e p a r a t i o n was placed in a R i n g e r ' s solution s u p p l e m e n t e d with 0.9 mM Co 2+.
A t the end of the incubation period, the nerves were d e s h e a t h e d and 4 m m segments
i m m e d i a t e l y proximal to the ligatures were saved for gel analysis. T h e r e m a i n d e r of
the p r e p a r a t i o n was cut into 3 m m segments t h a t were each placed in 5% trichloroacetic acid ( T C A ) overnight at 4°C. A f t e r the removal of T C A , nerve segments were
dissolved in N C S ( A m e r s h a m - S e a r l e ) , scintillant was a d d e d (4.95 g PPO, 0.05 g
P O P O P , 0.6 ml glacial acetic acid per liter toluene) and samples were counted in a
B e c k m a n L S 7 0 0 0 scintillation spectrometer. Efficiency of counting was 91%, as
d e t e r m i n e d with external s t a n d a r d s at a b a c k g r o u n d of 28 cpm.
Two-Dimensional
Gel
Electrophoresis
Modifications of the O ' F a r r e l l - t y p e gel system have been presented in detail
( W i l s o n et al., 1977; Stone et al., 1978). F o u r m m nerve segments were homogenized
(glass-glass) in 30 ~1 of a m i x t u r e of 1% S D S , 10% ~ - m e r c a p t o e t h a n o l , and 9.5 M
urea. To this h o m o g e n a t e was a d d e d 10 gl of a m i x t u r e of 10% N P - 4 0 , 8% 5 - 7
ampholines, 2% 3 - 1 0 ampholines ( v / v ) ( B i o - R a d ) . T h e homogenizer was rinsed twice
with 20 #1 each of O ' F a r r e l l ' s lysis buffer ( O ' F a r r e l l , 1975). S a m p l e s were then spun
at 100,000 × g for 60 rain (22°C) before loading the s u p e r n a t a n t on the first
dimension gel. Also, d u p l i c a t e 2 ~tl aliquots of the h o m o g e n a t e were a d d e d to 500 #1
10% T C A , and the p r e c i p i t a t e was counted to d e t e r m i n e the a m o u n t of radioactive
m a t e r i a l applied to the gel.
6
Stone and Hammerschlag
The first dimension isoelectric focusing tube gel (pH - 4.5 to 8.5) was run for 19
hr at 400 V. Ten percent SDS slab gels run at 18 mA separated proteins in the second
dimension according to molecular weight (-20,000 to 200,000 daltons). Following
SDS electrophoresis, the gels were fixed and stained with Coomassie blue, impregnated with PPO (Laskey and Mills, 1975) and dried prior to fluorographic exposure at
-70°C for up to 2 months.
Quantitation of Gel Spots
The procedure for quantitatilag selected spots from the gel follows that described
by Wilson et al, (1977). The positions of individual spots on the gel were located using
the fluorographic pattern as a guide. Since virtually none of the rapidly transported
species were present in enough abundance to be seen on the gel staining pattern (Stone
et al., 1978), the borders of the gel were used to align the fluorograph with the gel. The
position of selected transported spots were then marked by tracing with carbon paper.
These spots were cut from the dried gel with a scalpel and wiped free of carbon. Each
gel piece was soaked in scintillation fluid (4 g PPO, 0.025 g POPOP, 100 ml NCS, 20
ml of 4 M NH4OH per liter of toluene) with agitation for at least 30 hr prior to
counting. This method was found to remove >95% of radioactivity from the gel piece
(Wilson et al., 1977). Samples were recounted after 24 hr of additional agitation to
ensure that no further counts could be eluted. Since many of the protein spots
analyzed were found to contain as few as several counts above background, all samples
were counted for 40 min to reduce statistical variation. By this means 3 cpm above
background could be detected with a standard deviation of _+1 cpm. Efficiency of
counting was 93%, using external standards at a background of 28 cpm (Beckman
LS7000).
Data Analysis
A series of normalization procedures was performed on cpm values of each gel
spot to allow comparisons between preparations. First, values were corrected for
changes in [35S] half-life from the time that the labeled methionine was produced until
the radioactive protein in the gel spot was counted. In addition to the 48 gel spots
chosen for quantitation, a gel piece was taken to determine background radioactivity
of the gel.
Values representing total transported protein that accumulated proximal to
ligatures were corrected for differences in incorporation between experimental and
control paired preparations. Total radioactivity for a particular nerve was determined
by adding cpm from the DRG and all nerve segments proximal to the ligature of the
nerve, after subtracting estimates of local incorporation based on cpm present in
comparable segments distal to ligatures. Values from control and experimental nerves
(from the same animal) were compared to generate a correction factor. This factor
was applied to values for the 48 individual gel spots on the assumption that differences
in incorporation are reflected equally in all protein species.
The two-dimensional gel procedure involves uncontrollable losses of material
(O~Farrell, 1975; Stone et aI., 1978). Thus the percent inhibition of transport as
determined from homogenates of nerve segments proximal to ligatures was compared
Subpopulationsof Fast-TransportedProteins
7
to percent inhibition derived by comparing the sum of the 48 quantified spots taken
from each gel of a control/experimental pair. Differences in these percent values
reflect net losses that occur during the gel procedure. All 48 spots were corrected for
such losses for a particular gel pair.
Next, the corrected cpm value for each spot on an experimental gel was expressed
as a percent of the value for the corresponding control gel spot. Finally, to allow the
comparison of control/experimental pairs of gels from different preparations, these
percent values were normalized with respect to the mean percent value of all 48 spots
from that gel pair. The values following this series of corrections and comparisons
represent an index of relative inhibition. That is, values less than or greater than 1.0
correspond to species that are respectively less sensitive or more sensitive to Co 2+ than
the average of all spots analyzed on that gel pair.
RESULTS
Qualitative Effects of Co 2+ on Rapid Axonal Transport
Cobalt is known to inhibit the amount of fast transported protein, but not the rate
at which it is transported (Hammerschlag et al., 1976). In the present study, exposure
of the DRG to 0.9 mN Co 2+ resulted in 66_+6% (mean _+ SE, n = 8) inhibition of the
amount of radioactive protein that accumulated in the 4 mm segment proximal to
ligations of the spinal nerves (Table I). As also shown in Table I, TCA precipitable
cpm in the DRG are 106_+9% of control, indicating that this level of Co 2+ does not
inhibit protein synthesis. Rapidly transported species that accumulated at ligatures on
spinal nerves of control (Fig. I A) and Co2+-treated (Fig. 1B) preparations were
compared following two-dimensional gel electrophoresis. Both gel patterns show over
70 individual protein species and are reminiscent of patterns presented in previous
studies (Stone et al., 1978; Stone and Wilson, 1979). Co 2+ appears to have no major
qualitative effects on the rapidly transported proteins, since virtually all the proteins
transported in control preparations were consistently seen in the transport patterns
from Co2+-treated preparations.
Quantitative Effects of Co 2+ on Rapid Axonal Transport
The lack of qualitative differences in the gel pattern coupled with the overall
inhibition in amount of transport suggests that all rapidly transported proteins are
influenced by Co 2+. A more detailed analysis of the effects of Co 2+ was carried out by
a quantitative comparison of the 48 most abundant gel spots chosen from the
fluorographic pattern (Fig. 2). Some of the spots chosen for quantitation may
represent multiply charged forms of individual proteins. Based on their consistency in
the labeling pattern, as well as lack of such charged species in the associated gel
staining pattern, these charged species are thought not to be artifactual (Stone et al.,
1978).
Normalization of raw cpm of the chosen spots from eight control/experimental
pairs of gels resulted in a range of values for relative inhibition. Visual inspection
suggested that the most abundantly transported proteins of relatively low M.W.
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Stone and Hammerschlag
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Fig. Z. Traced reconstruction of a fluorographic pattern of rapidly transported proteins. The
pattern identifies the 48 most abundant transported species (filled symbols) that were chosen for
quantitative analysis. Those spots outlined by dashes were analyzed together, since they could not be
resolved on all gels as individual spots, m.w. = molecular weight separation. Molecular weight
determinations were made with bovine serum albumin (BSA), ovalbumin (OVA), chymotrypsinogen
(CHY), and myoglobin (MYO). pI = isoelectric point separation. The pI values at the bottom are
approximate.
(-20,000 to 35,000 daltons) were less affected by C o 2+ than species of higher M.W.
This observation prompted a comparative examination of the effect of Co 2+ on
individual selected spots above and below -35,000. Such an analysis yielded two
populations of proteins with apparent differential sensitivities to Co 2+ (Fig. 3). While
not completely distinct on the basis of M.W., the differential sensitivity appears
consistent. Two-tailed t statistics of the means of the two populations are highly
significant for the examples shown (p values range from <0.01 to <0.001). When all
eight pairs of normalized data are analyzed, this trend is conserved (mean + SE of low
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1.0
INHIBITION
1.2
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Fig. 3. Frequency histograms of quantitatively
analyzed gel spots. Relative inhibition values of 48
spots are shown for three representative gel pairs (A,
B, and C). Values (as defined in detail under Methods) either greater than or less than 1.0 reflect
proteins that are respectively more sensitive or less
sensitive to Co 2÷ than the average of all spots
analyzed on that control/experimental gel pair. Each
histogram also identifies proteins above (closed
symbols) and below (open symbols) approximately
35,000 daltons molecular weight. The two molecular
weigh-t populations are significantly different in all
three cases: A,p < 0.00l; B,p < 0.01; C , p < 0.01.
+~
+~ +
+I
+
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+I +
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o o o o o o [-- o o t-. ~
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12
Stone and Hammerschlag
molecular weight population - 0.88 _+ 0.02; high molecular weight population =
1.10 _+ 0.01). It is also apparent that the spots included in the lower M.W. population
are present in much greater abundance at the ligature than spots in the higher M.W.
subgroup. That is, the lower M.W. spots account for approximately 80% of label found
in all the spots analyzed on each gel.
While the frequency histograms suggest at least two populations of transported
proteins differentially inhibited by Co 2+, it is the reproducibility of the level of
inhibition for each spot that emphasizes the significance of the effect (Table II). Data
presented are relative inhibition values for the 48 spots on different pairs of gels. At all
levels of inhibition, responses to Co 2+ appear to be quite consistent. The listing has
been divided into two groups based on the populations shown in Fig. 3. There appear to
be several exceptions when M.W. is used as a sole criterion for distinguishing the two
populations of transported species. Three gel spots (indicated in Table II by an *)
placed initially according to M.W. were changed based on significance of differences
in their relative inhibition values compared to means of both M.W. populations (p <
0.01 for all three spots).
By combining data presented in Fig. 3 and Table II, a composite gel pattern was
derived to identify the two populations of transported species (Fig. 4). The spots
representing each population were chosen based on visual inspection of apparent
relative abundance, molecular weight, and consistency of relative inhibition for given
spots.
pl'
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mw
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c:~ ~:D~ I
9
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Fig. 4. Traced reconstruction identifying transported proteins differentially sensitive to Co 2+.
Those protein species relatively less sensitive to Co 2+ are indicated by hatched symbols, those more
sensitive are shown by filled symbols. The numbers identify corresponding spots according to relative
inhibition values shown in Table II. Three spots, numbers 1, 7, and 36, are seen to be exceptions to
using molecular weight as a sole criterion for identifying the two populations. Closely apposed spots
surrounded by dashed lines denote species that were analyzed together, pI = separation according to
charge; m.w. = molecular weight separation.
Subpopulationsof Fast-Transported Proteins
13
DISCUSSION
The observation that cobalt inhibits the amount of protein rapidly transported
from the dorsal root ganglion (Hammerschlag et al., 1976; Hammerschlag and
Lavoie, 1979) is substantiated in the present study. A more critical analysis of the
effect of cobalt has been performed using high-resolution two-dimensional gel electrophoresis. Qualitative analyses of individual rapidly transported proteins have shown
that Co 2+ markedly inhibits the amount of all protein species exported from neuronal
somata. This is reflected in the overall similarity of the two-dimensional gel patterns
despite decreases in abundance of transported species in Co 2+ preparations. These
findings strengthen the proposal that a CaE+-dependent, Co2+-sensitive step is present
at s,ome common point in the pathway taken by all rapidly transported proteins prior
to their translocation (Dravid and Hammerschlag, 1975b; Hammerschlag and Lavoie,
1979).
Quantitative studies of 48 spots from corresponding control and experimental
gels reinforce this conclusion. In each case, Co 2+ inhibited the amount of protein
exported from the neuronal soma. Despite a graded distribution of the relative
inhibitory values, visual inspection of fluorographic patterns suggested that rapidly
transported proteins could be divided into two populations. When spots were grouped
into M.W. categories above and below -35,000, the two populations showed statistically distinct sensitivities to Co 2+ when mean relative inhibitory values for each group
were calculated. The finding that proteins of each subgroup were consistently
inhibited to similar degrees adds significance to this observed differential effect of
Co 2+.
It should be emphasized that the choice of molecular weight as well as the 35,000
dalton cut-off point are somewhat arbitrary criteria, and do not rule out other
parameters that may be involved in intracellular sorting of proteins during initiation of
rapid transport. Indeed, several other criteria appear to support the existence of these
subpopulations of transported species. The majority of spots in the higher M.W. group
are observed as multiply charged forms. That is, some proteins are represented as a
series of spots of similar M.W. and evenly spaced in the isoelectric focusing dimension.
In contrast, the members of the lower M.W. population do not demonstrate such
obvious charge heterogeneity. One explanation for such differences concerns the
well-documented rapid transport of glycoproteins (Forman et al., 1971; Karlsson and
Sj6strand, 1971; Edstr~Sm and Mattsson, 1973; Droz et al., 1973). Since these
macromolecules can possess variously charged terminal glycomoieties, they would be
predicted to resolve into multiply charged species on the two-dimensional gels (Stone
et al., 1978). Should this be the case, the differences in expression of charge between
higher and lower M.W. populations may be the consequence of sorting of transported
proteins at the level of the Golgi apparatus, where terminal glycosylation occurs
(Schachter, 1974; Leblond and Bennett, 1977).
Of additional interest are differences in the relative abundance of the two
subpopulations. The 16 lower M.W. species that are relatively less sensitive to Co 2+
account for approximately 80% of the total radioactivity associated with the 48
analyzed spots. By contrast, the higher M.W. group contains 32 of the 48 spots, but
only 20% of the total radioactivity. Preliminary evidence suggests that similar
populations are also revealed when turnover of fast transported proteins in the
14
Stone and Hammerschlag
ganglion is examined. Pulse/chase experiments with dorsal root ganglia demonstrate
similar populations of transported proteins, which appear to exit the cell body with
little delay after synthesis (low M.W. subgroup), or accumulate in the soma prior to
export (higher M.W. subgroup) (Stone and Hammerschlag, in preparation). Future
studies will assess whether these differences in sensitivity to Co 2+, relative abundance,
terminal glycosylation, and somal processing may be related to differential destination
and function of the two subgroups of fast transported species. It will also be important
to determine further the criteria for defining these populations. Reliance mainly on
molecular weight, despite yielding statistically different mean values of relative
inhibition, results in a degree of overlap in the values of both groups.
The present findings can be interpreted to suggest that more than one Ca 2+dependent step occurs during the initiation of rapid transport. Thus the lower M.W.
population of transported proteins, less influenced by Co 2+, would be subject to only
one Ca 2+ step, while those proteins demonstrating a greater sensitivity to Co 2+ would
be routed through more than one Ca2+-requiring process. To identify the cellular basis
of Ca2+-dependent steps occurring during the initiation of rapid transport, a proposed
analogy to exocrine secretion is helpful (Hammerschlag and Lavoie, 1979). From
studies on secretory systems (Caro and Palade, 1964; Jamieson and Palade, 1967a,b,
1968 a,b; Novikoff et al., 1971; Novikoff, 1976; Novikoff et al., 1977; Tartakoff and
Vassalli, 1978), it appears that newly synthesized proteins destined for secretion are
associated with the endoplasmic reticulum (ER) membrane. By a process of
membrane flow, proteins are then transferred to the Golgi by means of transition
vesicles (Jamieson and Palade, 1967a,b). Morphological evidence in both Ca2+-free
(Tartakoff and Vassalli, 1978) and Co2+-treated (Kern and Kern, 1969) preparations
provide evidence to suggest that fusion of these vesicles to the forming face of the
Golgi is a Ca2+-mediated process. In both studies, the altered ionic conditions resulted
in a proliferation of membrane profiles in the region of pre-Golgi ER. During the
initiation of rapid axonal transport, a similar Ca2+-induced fusion may occur, since the
proliferation of pre-Golgi profiles has also been observed in DRG exposed to Co 2+
conditions that inhibit fast transport (Lindsey et al., 1980). An alternative view of
membrane flow is based on the observation of direct tubular connections between the
pre-Golgi smooth ER and Golgi cisternae (Teichberg and Holtzman, 1973; Morr6 et
al., 1974; Rambourg et al., 1974; 1979). Conceivably, the occurrence of vesicle fusion
as well as direct tubular connections could explain the differential effects of Co 2+ in
the present study. One population of proteins transferred to the Golgi via transition
vesicles would be subject to a CaZ+-dependent step, while those proteins conveyed
along continuous tubes would bypass this C a 2+ requirement.
A second Ca2+-dependent step in exocrine secretion is the well-documented
fusion of Golgi-derived vesicles to the plasma membrane (Douglas, 1968; Rubin,
1970; Palade, 1975). During the initiation of fast transport, a similar CaZ+-requiring
fusion has been proposed from experiments with labeled fucose. A dose-dependent
Co 2+ inhibition of the export of fucosylated proteins from DRG sensory neurons,
together with lack of any effect of Co 2+ on fucose incorporation or on glycoprotein
translocation along desheathed nerves, has provided evidence for a CaZ+-requiring
process during initiation, which occurs after proteins have left the Golgi (Hammerschlag and Lavoie, 1979). These findings have led to the proposal that the translocation phase of rapid transport begins when Golgi-derived vesicles participate in a
Subpopulations of Fast-TransportedProteins
15
Ca2+-induced fusion with the axonal smooth ER (Hammerschlag, 1980). Data from
the present study suggests that the analogy of initiation of rapid axonal transport to
secretory events can be expanded. While all fast-transported proteins may reach the
transport system via a common post-Golgi, Ca2+-dependent step, the pathway for a
subpopulation of these proteins also involves a second, pre-Golgi, Ca 2+ step. This
seems a likely interpretation, although one additional role of Ca 2+ in the secretory
process may be relevant to the observed effect of Co 2+ in the present study.
Golgi-derived vesicles have been shown to contain and release Ca 2+ together with
secretory proteins (Wallach and Schramm, 1971; Argent et al., 1973; Chandler and
Williams, 1974). Conceivably, such intravesicular stores of Ca 2+ may be involved in
the processing of fast transported proteins during initiation.
SUMMARY
1. Effects of cobalt on individual rapidly transported proteins have been examined. Bullfrog dorsal root ganglia were pulse-labeled in vitro in normal medium or
Ca2+-free medium supplemented with 0.9 mM Co 2+, followed by fast transport in
spinal nerves.
2. Labeled proteins present in the 4 mm segment immediately proximal to each
nerve ligature were subjected to two-dimensional gel etectrophoresis (M.W. - 20,000
to 200,000 daltons, pI - 4.5 to 8.5).
3. Gel fluorographic patterns of Co2+-treated preparations revealed no qualitative differences compared to control transport patterns.
4. A quantitative analysis of 48 spots revealed that the amount transported of
each of the species analyzed was inhibited by Co 2+.
5. The spots could be divided into two significantly different populations
( p < 0.01) differentially sensitive to Co 2+. One group of 16 spots, mostly of -20,000
to 35,000 daltons, was less inhibited by Co 2+ and comprised - 8 0 % of the label in all
the spots analyzed. The other group of 32 spots, all of higher molecular weight, were
more sensitive to Co 2+ and contained - 2 0 % of the label.
6. The fact that all proteins were affected by Co 2+ supports the proposal that fast
transported proteins are subject to a common Co2+-sensitive, Ca2+-requiring step.
Further, the identification of differential effects of Co 2+ on transported species
suggests that more than one CaZ+-dependent step occurs during the initiation of fast
transport.
ACKNOWLEDGMENTS
We thank Colleen Heublein for her expert secretarial assistance. The critical
comments of Drs. D. L. Wilson and G. W. Perry are also greatly appreciated.
REFERENCES
Argent, B. E., Case, R. M., and Scratcherd, T. (1973). Amylasesecretion by the perfusedcat pancreas in
relation to the secretion of calcium and other electrolytes and as influenced by external ionic
environment.J. Physiol 230:575 593.
16
Stone and Hammerschlag
Caro, L. G., and Palade, G. E. (1964). Protein synthesis, storage, and discharge in the pancreatic exocrine
cell. An autoradiographic study. J. Cell Biol. 20:473-495.
Chandler, D. E., and Williams, J. A. (1974). Pancreatic acinar cells: effects of lanthanum ions on amylase
release and calcium ion fluxes. J. Physiol. 243:831-846.
Douglas, W. W. (1968). Stimulus secretion coupling: the concept and clues from chromaffin and other cells.
Br. J. Pharmacol. 34:451-474.
Dravid, A. R., and Hammerschlag, R. (1975a). Axoplasmic transport of proteins in vitro in primary
afferent neurons of frog spinal cord: effect of CaE+-free incubation conditions. J. Neurochem.
24:711-718.
Dravid, A. R., and Hammerschlag, R. (1975b). The role of calcium in axonal transport: gel electrophoretic
comparison of proteins undergoing 'fast' transport in normal and calcium-free medium. Abstr. Int. Soc.
Neurochem. 5:261.
Droz, B. (1969). Protein metabolism in nerve cells. Int. Rev. Cytol. 25:363-390.
Droz, B. (1975). Synthetic machinery and axoplasmic transport: maintenance of neuronal connectivity. In
Brady, R. O., (ed.), The Nervous System Volume 1: The Basic Neurosciences, Raven Press, New
York, pp. 111-127.
Droz, B., Koenig, H. L., and Di Giamberardino, L. (1973). Axonal migration of protein and glycoprotein to
nerve endings. I. Radioautographic analysis of the renewal or protein in nerve endings of chicken
ciliary ganglion after intracerebral injection of [3H]lysine. Brain Res. 60:93 127.
Eagle, H. (1959), Amino acid metabolism in mammalian cell cultures. Science 130:432-437.
Edstr6m, A., and Mattsson, H. (1973). Electrophoretic characterization of leucine-, glucosamine- and
fucose-labelled proteins rapidly transported in frog sciatic nerve. J. Neurochem. 21:1499-1507.
Forman, D. S., McEwen, B. S., and Grafstein, B. (1971). Rapid transport of radioactivity in goldfish optic
nerve following injections of labelled glucosamine. Brain Res. 28: t 19-130.
Hammerschlag, R. (1980). The role of calcium in the initiation of fast axonal transport. Fed. Proc.,
39:2809-2814.
Hammerschlag, R., and Lavoie, P.-A. (1979). Initiation of fast axonal transport: involvement of calcium
during transfer of proteins from Golgi apparatus to the transport system. Neurosci. 4:1195 2001.
Hammerschlag, R., Dravid, A. R., and Chiu, A. Y. (1975). Mechanism of axonal transport: a proposed role
of calcium ions. Science 188:273 275.
Hammerschlag, R., Chiu, A: Y., and Dravid, A. R. (1976). Inhibition of fast axonal transport of
[3H]protein by cobalt ions. Brain Res. 114:353-358.
Jamieson, J. D., and Palade, G. E. (1967a). Intracellular transport of secretory proteins in pancreatic
exocrine cell. I. Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34:577 596.
Jamieson, J. D., and Palade, G. E. (1967b). Intracellular transport of secretory proteins in the pancreatic
exocrine cell. II. Transport of condensing vacuoles and zymogen granules. J. Cell Biol. 34:597 615.
Jamieson, J. D., and Palade, G. E. (1968a). Intracellular transport of secretory proteins in pancreatic
exocrine cell. llI. Dissociation of intracellular transport from protein synthesis. J. Cell Biol. 39:590588.
Jamieson, J. D., and Palade, G. E. (1968b). Intracellular transport of secretory proteins in pancreatic
exocrine cell. IV. Metabolic requirements. J. Cell Biol. 39:589-603.
Karlsson, J.-O., and Sj6strand, J. (1971). Rapid intracellular transport of fucose-containing glycoproteins
in retinal ganglion cells. J. Neuroehem. 18:2209-2216.
Kern, H. F., and Kern, D. (1969). Elektronenmikroskopische Untersuchungen uber die Wirkung yon
Kobaltchlorid auf das exokrine Pankreasgewebe des Meerschweinchens. Virchows Arch. 4:54-70.
Laskey, R. A., and Mills, A. D. (1975), Quantitative film detection of 3H and 14C in polyacrylamide gels by
fluorography. Eur. J. Biochem. 56:335-341.
Lavoie, P.-A., Bolen, F., and Hammerschlag, R. (1979). Divalent cation specificity of the calcium
requirement for fast transport of proteins in axons of desheathed nerves. J. Neurochem. 32:17451751.
Leblond, C. P., and Bennett, G. (1977). Role of Golgi apparatus in terminal glycosylation. In Brinkley,
B. R., and Porter, K. R. (eds.), International Cell Biology, Rockefeller University Press, New York,
pp. 326-336.
Lindsey, J. D., Hammerschlag, R., and Ellisman, M. H. (1980). An increase in smooth endoplasmie
reticulum and a decrease in Golgi apparatus occur with ionic conditions that block initiation of fast
axonal transport. Brain Res., 205:275-287.
Morr6, D. J., Keenan, T. W., and Huang, C. M. (1974). Membrane flow and differentiation: origin of Golgi
apparatus membranes from endoplasmic reticulum. Adv. Cytopharmacol. 2:107-125.
Novikoff, P. M., Novikoff, A. B., Quintana, N., and Hauw, J.-J. (1971). Golgi apparatus, GERL, and
lysosomes of neurons in rat dorsal root ganglia, studied by thick section and thin section cytochemistry.
J. Cell Biol. 50:859-886.
Novikoff, A. B. (1976). The endoplasmic reticulum: a cytochemist's view (a review). Proc. Natl. Acad. Sci.
USA 73:2781-2787.
Subpopulations of Fast-Transported Proteins
17
Novikoff, A. B., Mori, M., Quintana, N., and Yam, A. (1977). Studies of the secretory process in the
mammalian exocrine pancreas. I. The condensing vacuoles. J. Cell Biol. 75"148-165.
O'Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem.
250:4007 4021.
Palade, G. E. (1975). lntracellular aspects of the process of protein secretion. Science 189:347-357.
Rambourg, A., Clermont, Y., and Marraud, A. (1974). Three-dimensional structure of the osmiumimpregnated Golgi apparatus as seen in the high voltage electron microscope. Am. J. Anat. 140:2746.
Rambourg, A., Clermont, Y. and Hermo, L. (1979). Three-dimensional architecture of the Golgi apparatus
in Sertoli cells of the rat. Am. J. Anat. 154:455-476.
Rubin, R. P. (1970). The role of calcium in the release of neurotransmitter substances and hormones.
Pharmac. Rev. 22:389418.
Schachter, H. (1974). The subcellular sites of glycosylation. Biochem. Soc. Symp. 40:57-80.
Stone, G. C., and Hammerschlag, R. (1980). Effects of cobalt on rapid axonal transport: two-dimensional
gel analysis. Abstr. Amer. Soc. Neurochem. 11:143.
Stone, G. C., and Wilson, D. L. (1979). Qualitative analysis of proteins rapidly transported in ventral horn
motoneurons and bidirectionally from dorsal root ganglia. J. Neurobiol. 10:1-12.
Stone, G. C., Wilson, D. L., and Hall, M. E. (1978). Two-dimensional gel electrophoresis of proteins in
rapid axoplasmie transport. Brain Res. 144"287-302.
Tartakoff, A., and Vassalli, P. (1978). Comparative studies of intracellular transport of secretory proteins.
J. Cell Biol. 79:694-707.
Teichberg, S., and Holtzman, E. (1973). Axonal agranular reticulum and synaptic vesicles in cultured
embryonic chick sympathetic neurons. J. Cell Biol. 57:88-108.
Wallach, D., and Schramm, M. (1971). Calcium and the exportable protein in rat parotid gland. Eur. J.
Biochem. 21:433-437.
Wilson, D. L., Hall, M. E., Stone, G. C., and Rubin, R. W. (1977). Some improvements in two-dimensional
gel electrophoresis of proteins: protein mapping of eukaryotic tissue extracts. Anal. Biochem.
83:33-44.