Sea urchin sperm creatine kinase: The flagellar isozyme is a microtubule-associated protein

Experimental

Cell Research 178 (1988) 307-317

Sea Urchin Sperm Creatine Kinase: The Flagellar Is a Microtubule-Associated Protein ROBERT Departments

M. TOMBES, *,l A . FARR,? and BENNETT of *Biochemistry

and TBiological Seattle, Washington

Structure, 98195

Universiry

lsozyme

M. SHAPIRO*92 of Washington,

Sea urchin sperm contain two isozymes of creatine kinase (CrK) in the sperm head and tail, as termini of a phosphocreatine shuttle to transport energy. The head isozyme is located at the mitochondrion. By using an antibody prepared against denatured flagellar CrK, we now show that the tail isozyme exists along the entire flagellum. This unusual CrK isozyme, of M, 145 kDa, is a component of the flagellar axoneme as indicated by electron microscopic immunolocalization and cell fractionation. Flagellar CrK specifically reassociated with extracted sperm axonemes as well as with in vitro polymerized sea urchin egg microtubules. Neither sperm mitochondrial CrK nor mammalian muscle CrK bound to axonemes under similar conditions. Thus, although the two sperm isozymes have similar kinetic properties, they differ in affinity for microtubules, a characteristic that may determine the regional differentiation needed for establishing a phosphocreatine shuttle. 0 1988 Academic Press, Inc.

Creatine kinase (CrK),3 which interconverts ATP and phosphocreatine (PCr), has been proposed to buffer cellular ATP levels and/or mediate a PCr shuttle for energy transport [l]. For the latter role, two populations of CrK are necessary, at sites of energy production and consumption, respectively. Although separate CrK isozymes have long been known to exist in muscle, located at the mitochondrion and the sarcomere, direct functional evidence for such a shuttle is lacking

[Il. Recently, such evidence has been obtained for sea urchin sperm motility [2-51. Two isozymes of CrK exist in sea urchin sperm, located in the mitochondrion and tail [5]. Inhibition of CrK activity leads to paralysis of the distal sperm tail [4] and decreases respiration [2, 33 exactly as predicted from disruption of a PCr shuttle system. The sperm mitochondrial and tail CrK isozymes are distinct gene products with different structural properties and virtually indistinguishable kinetics [5]. The different structures of these isozymes may ensure that they are located at the appropriate termini of the sperm PCr shuttle. We have tested this hypothesis by ’ Current address: Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706. * To whom reprint requests should be addressed. ’ Abbreviations used: BSA, bovine serum albumin; Cr, creatine; CrK, creatine kinase; DTT, dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)ltetraacetic acid; FDNB, I-fluoro-2,4-dinitrobenzene; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; IgG, immunoglobulin G; MES, 2-(N-morpholino)ethanesulfonic acid; MFSW, Millipore-filtered seawater; N-AC, N-acetyl cysteine; NGS, normal goat serum; NP-40, Nonidet-P40; Octylglucoside, n-octyl, a-O-glucopyranoside; PAGE, polyacrylamide gel electrophoresis; PCr, phosphocreatine; P,, inorganic phosphate; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline. 307

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Tombes, Farr, and Shapiro

several means. A polyclonal antibody was prepared against the 145kDa flagellar isozyme of CrK that specifically reacted with the flagellar and not the mitochondrial isozyme. This antibody allowed a determination of the precise localization of the flagellar isozyme within the tail. In addition, certain reconstitution experiments have been developed to test the reassociation of flagellar CrK with extracted sperm axonemes or with heterologous microtubules. The results suggest that the 145kDa flagellar creatine kinase is a microtubule-associated protein.

MATERIALS

AND

METHODS

Sperm from Strongylocentrotus purpuratus were obtained and used to purify mitochondrial and flagellar isozymes of creatine kinase as previously described [5]. Mouse muscle creatine kinase was a gift of Dr. S. Hauschka; its properties have been extensively discussed (e.g., see [13] for references). Generation and characterization of antibodies against j7agellar CrK. Two female rabbits (New Zealand white) of less than 1 year of age were injected with SDS-polyacrylamide gel slices which contained the flagellar CrK of 145,ooO M,. Approximately 0.4 mg protein from the 0.5 M KC1 Procion Red peak of flagellar CrK activity (Ref. [5], Fig. 3, lane 8; IO-fold enrichment in specific activity over whole tails) was applied to a IS-mm-thick, preparative, 5-15% polyacrylamide gradient SDS gel. Electrophoresis was carried out three times the amount of time required for the dye front to reach the bottom of the gel. Upon Coomassie blue staining, the 145-kDa flagellar CrK was the primary band and was clearly separated from other proteins. Approximately 200 pg of flagellar CrK, estimated by comparison to molecular weight standards, was excised from the stained gel and then crushed in Tris buffered saline (TBS) to prepare a slurry (1.5 ml). This was mixed with 1.5 ml Freund’s complete adjuvant and sonicated on ice for two 30-s pulses. The sonicated mixture was extruded through a 23gauge needle three times. A total of 1.3 ml containing approximately 80 pg CrK was injected using a 22-gauge needle in six to eight sites along the back of each rabbit. Booster injections at 25 and 45 days after the primary injection used 25 pg flagellar CrK. Rabbits were bled (50 ml) 10 days after each booster injection. Serum was fractionated by ammonium sulfate precipitation and DEAE-Sephacel to purify IgG, as previously described [6]. This IO-fold concentrated IgG fraction was stored at -70°C and used in all experiments unless otherwise stated. lmmunoblots of SDS gels were used to examine the approximate titer and specificity of antiserum, as previously described [5]. Attempts at enzyme inactivation or immunoprecipitation were carried out with 2 pg/ml purified flagellar CrK in TBS, 1 mM EGTA, 0.4-l % NP-40, and 0.1% BSA. The effect of antisera on flagellar CrK activity was monitored after incubation for up to 12 h on ice by coupled spectrophotometric assay in the reverse direction [5]; activity losses were
kinase

isozymes.

Flagellar

creatine kinase

309

experiments, this extract was dialyzed for 8h into at least 100 vol of axoneme buffer lacking detergent. Egg microtubules were prepared as previously described [8], except for the following modifications. Eggs were dejellied by acidifying 15 ml of a 50% suspension of eggs in Millipore-filtered seawater (MFSW) with 0.15 ml 1.OM MES acid for 10 min followed by neutralization with 0.15 ml 1.O M Tiis base. Eggs were then hand centrifuged and the pellet (3 ml) was washed, homogenized, and extracted, and microtubules were polymerized with taxol [S]. SDS-PAGE of axonemes and egg microtubules revealed polypeptide profiles similar to those previously published with tubulin being the major protein component [5, 81. Reassociation experiments were performed by incubating axonemes or egg microtubules with sperm extracts or purified CrK in 50-100 ~1 axoneme buffer at 10°C with agitation on a rotary shaker. No difference in binding was observed when the incubation time was varied between 30 min and 3 h. The incubation was terminated by centrifuging the sample through 0.5 ml 15 % sucrose onto 0.5 ml of a 50% sucrose cushion in axoneme buffer, for 15 min at 10,OOOg(12,000 rpm in Sorvall HB-4). CrK activity was routinely measured in the sample prior to centrifugation and in the resuspended pellet after centrifugation to determine the percentage of activity that had bound to the microtubules. In some experiments, the activity in the supematant was determined to calculate the overall yield of CrK activity. which averaged 88%4120/o (n=15). lmmunolocalization of sperm isozymes: Light microscopy. Sperm were settled onto 0.1% poly-Llysme-coated coverslips for 30 min (20°C) then fixed in either 100% methanol at -20°C for 7 min or 3% paraformaldehyde in MFSW, pH 8.0, for 1 h (10°C). The subsequent incubations with antiflagellar CrK and TRITC-goat anti-rabbit IgG, mounting, and observations were as previously described [6]. .&Yiecfronmicroscopy. Sperm were settled onto poly-L-lysine-coated coverslips in MFSW and fixed in 3 % paraformaldehyde/MFSW, the fixative that best preserved morphology and antigenicity. Fixed, adhered sperm were then labeled with preimmune serum or anti-tail CrK, using TBS containing 1% BSA as the blocking buffer. The secondary antibodies were goat anti-rabbit IgG conjugated to horseradish peroxidase, or protein A conjugated to 10 nm gold. Sperm were stained for peroxidase using 0.5 mM 3,3’-diaminobenzidine and 0.004 % hydrogen peroxide, treated with 2% OsOd and then dehydrated. embedded in plastic, sectioned, and examined as described previously [9].

RESULTS Characterization

of Antisera

As shown in Fig. 1, the immune serum specifically reacted with purified flagellar CrK (lane 4), and flagellar CrK in whole sperm (lane 3) and in isolated tail preparations (lane 2). The specificity was confirmed by performing electrophoresis for an extended period of time followed by immunoblotting, in order to resolve other proteins that might have comigrated with flagellar CrK. Immunoreactivity was always associated with creatine kinase. Moreover, two-dimensional gel electrophoresis of the purified membrane fraction used as antigen demonstrated the absence of any proteins of a size similar to that of the flagellar isozyme in that fraction. There was no reaction with preimmune serum. The IgG fractions used here corresponded to serum dilutions of approximately 1000-fold, at which level there was no reaction with sperm heads (lane 2). The antibody reacted only weakly with mitochondrial CrK (lane 4) and did not react with mammalian CrK-MM at this concentration (lane 5), although cross-reactivity (with both isozymes) was seen with higher concentrations of antibody. Neither the anti-flagellar CrK undiluted antisera nor the concentrated IgG fractions were capable of precipitating or inactivating native tail CrK. However, when purified flagellar CrK was labeled with 3H-FDNB, and then heat and SDS

310

Tombes, Farr, and Shapiro

Protein 12345

a -CrK 12345

Fig. 1. Immunoblot of sperm proteins after SDS-PAGE. Proteins were stained with 0.1% amido black; immunostaining employed 40 &ml DEAE-purified anti-flagellar CrK IgG (corresponding to an approximately 250-fold dilution of pure serum). Lane I, sperm tails, 15 ug; lane 2, sperm heads, 8 ug; lane 3, whole sperm, 8 ug; lane 4, flagellar CrK, 1.2 ug, and mitochondrial CrK, 0.5 ug; lane 5, CrK-MM, 0.3 pg. Dashes represent, from top to bottom, molecular weight markers of 200, 116, 94, 68, 45, 31, 21, and 14 KDa. The arrow indicates the migration position of flagellar CrK.

denatured, the antisera precipitated flagellar CrK in the presence of protein A as confirmed by fluorography of SDS gels. At 2 l&ml labeled and denatured CrK, a maximum of 70% of the cpm were precipitated with a 1 : 2 or a 1 : 20 dilution of antiserum. These data are consistent with the fact that denatured CrK was used as immunizing antigen. Immunolocalization

of Sperm Isozymes

Immunostaining of methanol-fixed sperm with anti-flagellar CrK antibodies revealed staining along the entire length of the axoneme. Both the distal filament of the tail (Fig. 2, small arrow) and the centriolar region, which extends up into the base of the head, were stained (Fig. 2, large arrow). This staining pattern was also observed when sperm were fixed with aldehydes (not shown). There was no staining elsewhere in the sperm head (Fig. 2), although at approximately 5-fold higher levels of serum, very weak mitochondrial staining was seen. Preimmune serum, which showed no reaction at the levels used here, began to show staining of the centriolar region and acrosomal vesicle at 20-fold higher concentrations. Immunoelectron microscopy localized CrK uniformly and exclusively along

Flagellar

Fig. 2. Fluorescent immunolocalization of flagellar scribed under Materials and Methods and stained with CrK IgG (C, D) or preimmune IgG (A, B) followed by Images were photographed using either bright field (A, 10 urn. In (D), the long arrow points to light centriolar filament staining.

creatine kinase

3 11

CrK. Sperm were fixed in methanol as de100 ug/ml of either DEAE-purified antitlagellar goat anti-rabbit IgG conjugated to rhodamine. C) or fluorescence (B. D) optics. Scale bar = staining and the shorter arrow points to distal

the flagellum. CrK was found on either edge of the flagellum and in a central line passing down the middle of the axoneme when sectioned longitudinally (Fig. 3B). This staining pattern was identical to the electron density pattern of the “demembranated” sea urchin sperm flagellum (e.g., see [lo]). In cross-section, staining was in an outer circle as well as in the middle of the axoneme (Fig. 3 0, in the pattern of electron density that is attributed to the 9+2 arrangement of microtubules of the axoneme. Mitochondria were not stained, although they were stained with antibodies against CrK-MM [5]. Preimmune serum did not show any reaction at the concentrations of antibody used in these EM studies (Fig. 3A). Flagellar

CrK in Isolated

Axonemes

and Membranes

CrK activity was examined in fractions from standard preparations of both flagellar membranes and axonemes. Membranes prepared as previously de21-888340

Fig. 3. Immunoelectron microscopic localization of flagellar CrK. DEAE-purified preimmune IgG and anti-flagellar CrK IgG (B. C) at 100 &ml (1 : 100 dilution) were used to stain 3 % paraformaldehyde-fixed and 0.05% NP-40-washed sperm. They were then reacted with goat anti-rabbit IgG conjugated to horseradish peroxidase and developed with 3,3’-diamino benzidine (DAB). Reacted sperm were then dehydrated, embedded, and sectioned for transmission electron microscopy as described in the text. Scale bar = 1 urn. (A)

scribed [ 111 exhibited only a small amount of CrK activity and did not show the increase in specific activity that the Na+/K+ ATPase did in the same preparation. This low level of CrK activity was attributed to the flagellar isozyme by immunoblotting (data not shown). Membranes prepared according to other procedures [121, however, did not contain this protein by immunoblotting, suggesting that association of CrK with membranes in certain preparations may be adventitious. Likewise, ony a small amount of CrK remained with axonemes prepared by standard detergent-containing procedures [7] at 5 mg/ml protein. The detergent concentrations required to half-maximally extract CrK from axonemes under these conditions were 0.025 % NP-40 and 0.75 % octylglucoside (data not shown). No combination of lower levels of detergent followed by salt treatment could extract as much activity as with detergent alone. Detergents might have removed CrK either by permeabilizing the flagellar membrane sufficiently to allow CrK to diffuse out or by disrupting a hydrophobic bond between CrK and the axoneme. The latter possibility was supported by the following CrK-axoneme reassociation experiments.

Flagellar

TABLE Axoneme

creatine kinase

3 I3

1

and egg microtubule

CrK binding

Source of CrK

% of input activity or counts bound (-background)

Undialyzed axonemal extract Dialyzed axonemal extract Purified flagellar CrK Pure CrK-MM Pure mitochondrial CrK ‘H-FDNB-Labeled CrK

20.4k7.9 (n=l3) 4.4kl.O (n=2) 3.8f2.3 (n=7) 1.0+0.7 (n=7) 0.5+0.2 (n=5) 6.8f 1.o (n=2)

(I) Undialyzed axonemal extract (2) Purified flagellar CrK (3) Pure CrK-MM

10.9t2.7 (n=6) 1.1+0.5 (n=3) (n=l) 0.05

A. Axonemes (1) (2) (3) (4) (5) (6) B. Taxol-stabilized egg micro tubules

Note. Association of CrK activity with axonemes and egg microtubules. Axonemes or microtubules (65-150 ug) were incubated with 200-2000 mU of CrK activity in the form of purified enzymes or crude extracts. The percentage of this input activity that remained bound to axonemes or microtubules after centrifugation through 15% sucrose is presented with standard deviations and sample numbers (n). Background activity represented the percentage of input activity that was found in the pellet after centrifugation in the absence of microtubules and never exceeded 1.0%.

Axoneme-CrK

Reassociation

Axonemes were routinely prepared by extracting tails three times at 5 mg/ml in 2.5% octylglucoside to ensure complete removal of CrK. Octylglucoside was used because it could be dialyzed away from the extract. All fractions or extracts which contained flagellar CrK exhibited reassociation of a significant fraction of the added activity when incubated with these axonemes (Table 1). This fraction of reassociated activity, expressed in terms of the percentage of the amount of input activity bound, varied greatly from an average of 20% with undialyzed extracts to around 4% in dialyzed extracts (Table 1). The percentage binding of the dialyzed extract (4.4%) was similar to the value for puritied flagellar CrK (3.8%). Although low, this binding was significant, specific, and typical of two different flagellar CrK preparations. Neither CrK-MM nor sperm mitochondrial CrK bound to axonemes at significant levels. Half-maximal competition of flagellar CrK binding by BSA or IgG occurred only at a 500-fold excess of the competing proteins (data not shown). Binding of CrK activity from tails was saturable with increased amounts of both axonemes and CrK (Fig. 4). When the undialyzed extract was diluted to 0.2% or less octylglucoside and added to a serial dilution of axonemes, binding began to saturate above 62 ug axonemal protein (Fig. 4A). When 62 ug of axonemes was used to test the binding of various levels of the dialyzed extract, saturation was also observed, showing a maximum binding of 4% of the input activity (Fig. 4B). Immunofluorescent

314

Tombes, Fart-, and Shapiro

0

100

200

300

pug Axonemes 8

2ooy

.YB

u 5 $ M

q 100

q

.E 3 E

:/

.?/!

I 0

2000

,

,

4000

6000

mUnits

Input

.

,

6000

11 IO0

Fig. 4. Saturation binding of CrK to axonemes. (A) Sperm axonemes were added to 1600 mU of an undialyzed sperm tail extract, incubated for 50 min, centrifuged, and analyzed as described under Materials and Methods. The final octylglucoside concentration was 0.2%. (B) The indicated levels of activity of a dialyzed sperm tail extract were added to 64 pg of axonemal protein and the amount bound was determined as in (A). The final octylglucoside concentration was GO.01 %.

staining of these reassociated axonemes indicated a uniform distribution of flagellar CrK (data not shown). The decrease in binding efficiency upon dialysis suggests that either flagellar CrK reassociation with axonemes is mediated by a dialyzable factor or that dialysis in detergent-free buffer results in some change in CrK that diminishes binding. We tested these possibilities in the following ways. First, the binding efficiency of dialyzed extracts or purified CrK was determined in the presence of undialyzed CrK extracts, which had been boiled for 3 min to inactivate their endogenous CrK activity, in the event that a heat-stable dialyzable binding factor existed. Binding efficiencies were not augmented in these mixing experiments. One potential heat-stable, dialyzable factor, CaC12, tested at 5 mM could not augment binding, but we tested no other ions. Fractions from the Procion Red columns (used to purify CrK) [5] that lacked CrK activity were also incapable of augmenting binding. These results suggest that the loss of binding was due to an irreversible alteration in flagellar CrK that happened when detergent was dialyzed away, but which did not affect the catalytic activity. Binding efficiencies also decreased as undialyzed CrK extracts were aged. In initial samples, the binding was as high as 40%, but decreased to 10% or less over a period of a few days, during which time the catalytic activity was stable.

Flagellar

creatine kinase

.:

NaCl

n :

OG

.:

NP-40

3 15

% Detergent or M NaCl 120 looa-& .

.:

\

60

q

NaCl

l : OG

.

40 _20

\

,\ \

0, 0.0

0.5 % Detergent

1.0

1. 5

or M NaCl

Fig. 5. Characteristics of CrK binding. (A) 2.3 ug (250 mu) of pure flagellar CrK or 250 mU of fresh flagellar extract was incubated with 62 ug axonemes in the presence of various concentrations of NaCI, octylglucoside, or NP-40. The percentage of the initial activity added that remained bound to centrifuged axonemes is presented as a percentage of the maximum binding in order to directly compare the binding of purified flagellar CrK with fresh flagellar CrK extract. The maximum percentage binding was 4.1% for pure flagellar CrK and 22 % for flagellar extracts in this experiment. NP-40 data were determined using fresh extracts; octylglucoside data used purified CrK; and NaCl data were obtained from both fresh extracts and purified CrK. Data for this figure were compiled from two separate experiments. (B) 200 mU of CrK extract in which the octylglucoside concentration had been diluted to 0.22 % was added to egg microtubules with increasing concentrations of octylglucoside or NaCl, incubated and centrifuged as described under Materials and Methods. The maximum percentage binding was 11.1%.

Fresh axonemes could not restore the initial level of binding activity. These data support the notion that the microtubule binding domain is more labile than the catalytic activity of flagellar CrK. Binding was completely disrupted by both octylglucoside and NP-40 and was relatively resistant to increased ionic strength (Fig. 5). CrK was incubated with axonemes in the presence of various concentrations of NaCl or detergents and its binding effkiency was determined. We have presented the data as a proportion of the initial binding efficiency so that data from all experiments can be compared. Octylglucoside at 0.55 % and NP-40 at ~0.2 % half-maximally prevented binding (Fig. 5A). NaCl, on the other hand, disrupted binding half-maximally at 1.0 A4 (Fig. 5A). This suggests that CrK axonemal binding is primarily hydrophobic in nature, even though this protein has properties typical of soluble proteins [5]. Egg Microtubules-CrK

Reassociation

Microtubules can be polymerized from sea urchin egg extracts with the microtubule stabilizing agent, taxol [8]. Sperm flagellar CrK bound to these microtu-

3 16 Tombes, Farr, and Shapiro

bules as it did to sperm axonemes, with CrK in fresh extracts binding to a greater degree (10.9%) than purified flagellar CrK (1.1%) (Fig. 4). We did not test whether other CrK isozymes or dialyzed tail extracts could reassociate with egg microtubules. The nature of the binding of CrK in fresh extracts to egg microtubules, however, also appeared to be primarily hydrophobic in nature, even though we were able to completely extract CrK at high salt concentrations (Fig. 5B). The polypeptide profiles of axonemes and egg microtubules were similar to each other, with tubulin representing the major protein in both cases (data not shown). DISCUSSION PCr shuttles facilitate energy transport between sites of energy production and consumption and can be identified by the existence of separate isozymes at each terminus [ 11. In sea urchin sperm, isozymes exist at the mitochondrion and tail [5] to mediate energy transport for motility [2A]. The flagellar terminus constitutes the most unusual CrK isozyme yet described [5] in an enzyme family that is otherwise highly conserved [13]. It possesses a IV, roughly three times that of conventional CrK monomers. We have now tested whether this large isozyme may possess specific localization information and shown that this unusual CrK isozyme colocalizes with the microtubules of the axoneme and possesses microtubule-associating properties in uitro, with both extracted sperm axonemes and in vitro polymerized egg microtubules. Flagellar CrK immunostaining is uniform along the entire flagellum extending from the centriole to the distal filament (Fig. 2). This centriolar staining is consistent with a microtubule rather than a membrane association, as is the similarity of EM immunostaining patterns of CrK with the axonemal microtubule array (Fig. 3). The uniformity of staining along the flagellum suggests that there is no extreme gradient of CrK molecules along the tail to regulate facilitated energy transport. This result supports the assumption used in our model for CrKfacilitated energy transport, that CrK is distributed uniformly along the flagellum [41.

CrK binding to extracted axonemes is saturable with both axonemes and CrK, thus implying a specific binding site. However, although purified CrK binds specifically and significantly to axonemes, it binds with low efficiency, while binding efficiencies of flagellar CrK in fresh extracts can be as high as 40 % of the added activity. This irreversible loss of binding efficiency seems to be caused by either sample aging or detergent removal, rather than by the loss of a binding factor, although this point will have to be substantiated with more direct experiments. The specificity of the localization of the two sperm CrK isozymes to the mitochondrion and the flagellum is shown from these data and a previous immunological study [5]. It is particularly emphasized by noting the exclusive localization of the two isozymes at the ambiguous head/tail junction where the centriole, which is embedded in the head, is surrounded by the donut-shaped

Flagellar

creatine kinase

3 17

mitochondrion. The flagellar CrK is found only in the centriole (Fig. 2) while the mitochondrial CrK resides only in the mitochondrion [5]. The specificity of subcellular localization of the isozymes was further supported by the in vitro microtubule reassociation experiments in which flagellar CrK, but neither mitochondrial CrK nor a mammalian CrK isozyme reassociated with extracted sperm axonemes. The binding of flagellar CrK is not restricted to microtubules of the sperm axoneme, but occurs on microtubules polymerized with taxol from sea urchin eggs as well. Although we have not tested its binding to microtubules polymerized from pure tubulin, the fact that CrK binds to egg microtubules, even though CrK is virtually undetectable in sea urchin eggs and early embryos [14, Tombes, unpublished observations], also suggests that binding of CrK to microtubules is direct and is not mediated by a specific CrK binding factor. Although an extra domain(s) of this 145-KDa flagellar CrK may be involved in its axonemal association, this is not obligatory for CrK localization, since mammalian CrK isozymes of 45 KDa have the information needed for their particular subcellular locations [15]. Nonetheless, our data suggests that structural differences between sea urchin isozymes ensure that each isozyme is placed at its respective terminus of the PCr shuttle during spermatogenesis. Current research is directed toward elucidating the primary structure of this unique flagellar CrK isozyme in order to define, in conjunction with the microtubule binding assay, regions that are responsible for microtubule association and catalytic activity. We acknowledge Jim Trimmer and Prapapom Toowicharanont for supplying sperm plasma membrane samples, Charles Rauch for taxol, and Susan K. Anderson for expert EM technical assistance. We also thank Mary Patella and Maureen Shea for excellent secretarial assistance and Mary Beth Tombes for useful suggestions. Supported by National Institutes of Health Grant GM 23910.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Bessman, S. P., and Carpenter, C. L. (1985) Annu. Rev. Biochem. 54, 831-862. Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334. Shapiro, B. M., and Tombes, R. M. (1985) Bioessyvs 3, 101-103. Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys, J. 52, 75-86. Tombes, R. M., and Shapiro, B. M. (1987) J. Viol. Chem. 262, 16,011-16,019. Gundersen, G. G., and Shapiro, B. M. (1984) Biochim. Biophys. Acta 799, 68-79. Gibbons, I. R., and Fronk, E. (1979) J. Biol. Chem. 254, 187-196. Vallee, R. B., and Bloom, G. S. (1983) Proc. Nat/. Acad. Sci. USA 80, 6259-6263. Farr, A. G., and Nakane, P. K. (1981) J. Immunol. Methods 47, 129-144. Sale, W. J. (1986) Cell Biol. 102, 2042-2052. Kazazoglou, T., Schackmann, R. W., Fosset, M., and Shapiro, B. M. (1985) Proc. Natl. Acad. Sci. USA 82, 1460-1464. 12. Podell, S. B., Moy, G. W., and Vacquier, V. D. (1984) Biochim. Biophys. Acta 778, 25-37. 13. Buskin, J. N., Jaynes, J. B., Chamberlain, J. S., and Hauschka, S. D. (1985) J. Mol. Euol. 22, 33&341.

14. Fujimaki, H., and Yanagisawa, T. (1978) Deu. Growth Differ. 20, 125-131. 15. Hall, N., and DeLuca, M. (1980) Arch. Biochem. Biophys. 201, 674-677. Received December 29, 1987 Revised version received April 11, 1988

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