(Received for publication, June 14, 1995; and in revised form, August 16, 1995)
From the
In mammalian spermatozoa, most of the type II isoform of
cAMP-dependent protein kinase (PKAII
) is anchored at the
cytoplasmic surface of a specialized array of mitochondria in the
flagellar cytoskeleton. This places the catalytic subunits of
PKAII
in proximity with potential target substrates in the
cytoskeleton. The mechanism by which PKAII
is anchored at the
outer surface of germ cell mitochondria has not been elucidated. We now
report the cloning of a cDNA that encodes a novel, germ cell A kinase
anchor protein (AKAP) designated S-AKAP84. S-AKAP84 comprises 593 amino
acids and contains a centrally located domain that avidly binds
regulatory subunits (RII
and RII
) of PKAII
and
PKAII
. The 3.2-kilobase S-AKAP84 mRNA and the cognate S-AKAP84 RII
binding protein are expressed principally in the male germ cell
lineage. Expression of S-AKAP84 is tightly regulated during
development. The protein accumulates as spermatids undergo nuclear
condensation and tail elongation. The timing of S-AKAP84 expression is
correlated with the de novo accumulation of RII
and
RII
subunits and the migration of mitochondria from the cytoplasm
(round spermatids) to the cytoskeleton (midpiece in elongating
spermatids). Residues 1-30 at the NH
terminus of
S-AKAP84 constitute a putative signal/anchor sequence that may target
the protein to the outer mitochondrial membrane. Immunofluorescence
analysis demonstrated that S-AKAP84 is co-localized with mitochondria
in the flagellum.
Multiple isoforms of cAMP-dependent protein kinase (PKA) ()are expressed during mammalian
spermatogenesis(1, 2, 3) . PKA isoforms are
generated by the synthesis of four types of cAMP-binding or regulatory
(R) subunits that are encoded by distinct
genes(4, 5) . R subunits undergo homodimerization and
confer unique physical and functional properties on heterotetrameric
(R
C
) PKAs that contain similar or identical
catalytic (C) subunits (reviewed in (4) and (5) ).
Individual PKA isoforms, which are named according to their R subunits,
are thought to play specialized physiological roles at certain stages
of male germ cell differentiation(1, 2, 3) .
Both the temporal patterns of expression and distinct intracellular
locations of R subunit isoforms are consistent with this idea. RI
and its cognate mRNA are expressed in premeiotic and early postmeiotic
germ cells(1, 2) . Moreover, the RI
polypeptide
persists in elongating spermatids and mature sperm. In contrast,
RII
mRNA and protein are initially expressed at a relatively late
postmeiotic stage. As spermatocytes proceed through nuclear
condensation and elongation the level of RII
polypeptide declines,
and this isoform is not detected in mature
sperm(1, 2) . RII
expression is initiated in
parallel with RII
accumulation. However, the level of RII
polypeptide becomes maximal in late spermiogenesis, and the protein
persists in mature, epididymal
sperm(1, 2, 3, 6) .
Activation of
PKA in mature spermatozoa elicits the initiation and maintenance of
flagellar movement(7, 8) . This well established
physiological response to cAMP is correlated with the cyclic
nucleotide-stimulated sliding of a cytoskeletal structure (the fibrous
sheath) over an internal microtubular structure called the
axoneme(9) . In mature spermatozoa PKAI
(RI
C
) is evident in the cytoplasm and
plasma membrane(3) . In contrast, a high proportion of RII
(and PKAII
) is sequestered in the cytoskeleton. Thus, the C
subunit of PKAII
may catalyze selectively the phosphorylation of
proximal regulatory and/or structural proteins in the cytoskeleton.
These phosphoproteins may, in turn, mediate the dynamic reorganization
of the flagellar cytoskeleton that is essential for motility and
fertilization.
Type II and type II
PKAs are often attached
to the cytoskeleton or other organelles via the binding of RII
or
RII
with A kinase anchor proteins (AKAPs) (reviewed in (10) and (11) ). AKAPs, as typified by neuronal AKAPs
75, 79, and 150(12, 13, 14) , are proteins
that possess both a high affinity binding site for RII
/RII
and an independent domain that targets and links RII-AKAP complexes to
specific intracellular sites(15) . This arrangement can place
PKAII
and/or PKAII
in proximity with organelle-bound
substrates, thereby creating a target site for cAMP
action(10, 11, 12, 13, 14) .
RII
(PKAII
) accumulates at two sites in the sperm
flagellum(16) . The bulk of the RII
polypeptide is
attached to the outer membrane of specialized ``germ cell''
mitochondria that form a helical array around the outer dense fibers
and axoneme in the ``midpiece'' of the flagellum. A lesser
fraction of RII
is associated with the fibrous sheath in a distal
portion (the ``principal piece'') of the flagellum.
Recently, Carrera et al.(17) cloned and sequenced
cDNAs encoding a major fibrous sheath protein p82. p82 binds RII
and contains two short segments that share sequence similarity with the
targeting domains of neuronal AKAP75(15) . It is likely that
p82 mediates the binding of PKAII
to the fibrous sheath.
Major
questions regarding the targeting of signals carried by cAMP in germ
cells remain unanswered. How is the bulk of PKAII anchored on the
outside surface of mitochondria? Does an AKAP mediate this
localization? If so, is a previously characterized or a novel AKAP
polypeptide used? Is the anchor protein developmentally regulated and
targeted principally to mitochondria?
We now describe the cloning and characterization of cDNAs that encode a unique germ cell anchor protein designated spermatid AKAP84 (S-AKAP84). S-AKAP84 contains an RII binding site and a putative mitochondrial targeting sequence. Moreover, S-AKAP84 accumulates as spermatids undergo nuclear condensation and flagellar elongation. The timing of expression of S-AKAP84 appears to be synchronized with the accumulation of RII subunits and the migration of mitochondria from the cytoplasm (round spermatids) to the cytoskeleton (midpiece in elongating spermatids). Immunofluorescence analysis documented directly that the RII binding protein is co-localized with the helical array of mitochondria in the midpiece of the flagellum.
Figure 1:
cDNA
and derived amino acid sequences for S-AKAP84. A, numbering
for the nucleotide sequence is presented on the right; amino
acid residue positions are indicated on the left. Panel B shows the locations and sequences of the candidate mitochondrial
signal/anchor domain, the potential leucine zipper region, and the
predicted RII/RII
binding site.
In recombinant pET14b, the partial S-AKAP84 cDNA is
preceded by plasmid DNA that encodes an initiator ATG and 19 additional
amino acids (including 6 consecutive His residues). Transcription of
the chimeric gene is governed by a promoter sequence for bacteriophage
T7 RNA polymerase. The partial S-AKAP84 protein (designated P21 in
accord with its calculated M of 21,000) was
expressed in Escherichia coli BL21 (DE3) and purified to near
homogeneity by metal-chelate affinity chromatography as described by Li
and Rubin(22) . A 500-ml culture of bacteria yielded 2 mg of
P21.
A multiple-tissue RNA blot containing
size-fractionated human poly(A) RNAs (2 µg/lane)
was obtained from Clontech. The blot was probed with the
P-labeled cDNA described above. Hybridization, washing,
and autoradiography were performed as described
previously(28) .
Subsequently,
a 499-base pair ApaI fragment, corresponding to nucleotides
123-621 at the 5` end of the T1 cDNA, was used to generate
P-labeled cDNA for further screening. A human testis cDNA
library enriched in 5` sequences yielded eight cDNA clones that were
analyzed by sequencing. The cDNA designated pGT7 contained novel
5`-coding and 5`-untranslated sequences linked to the complete
downstream coding and untranslated sequences determined for
T1
cDNA. The remaining phage clones contained overlapping partial cDNA
sequences that were identical to portions of the cDNAs in pGT7 and
T1. The net result from multiple rounds of analysis is that each
segment of the testis AKAP cDNA was sequenced from both strands in at
least three independent cDNA clones. The data are presented in Fig. 1A.
An open reading frame of 1779 base pairs begins at an initiator ATG (nucleotides 63-65) that lies within a consensus translation start site(29) . Although the exact size of the 5`-untranslated region is unknown, the occurrence of an in-frame translation stop codon at nucleotides 42-44 precludes the possible utilization of a distal, upstream initiator ATG. The AKAP open reading frame terminates with a stop codon at nucleotides 1842-1844 (Fig. 1A). The sequence of >500 base pairs of 3`-untranslated sequence was determined, but neither a poly(A) tail nor a polyadenylation signal was evident. Since a 3.2-kb mRNA hybridized with the newly cloned cDNA (see Fig. 4), it is possible that the total length of the 3`-untranslated region exceeds 1.2 kb. However, the length and complete sequence of the 5`-untranslated region also remain to be determined.
Figure 4:
Size
and abundance of S-AKAP84 mRNA in human tissues. A Northern blot, which
contained 2 µg of size-fractionated poly(A) RNA in
each lane, was probed with
P-labeled S-AKAP84 cDNA
corresponding to nucleotides 762-1260 in Fig. 1A (see
``Experimental Procedures''). The source of the
poly(A
) RNA is indicated at the top of each lane. The gel was calibrated by running an RNA ladder in a
parallel lane and staining with ethidium bromide. The sizes of the
major S-AKAP84 mRNA in testis and the minor S-AKAP84 transcript in
other tissues are indicated. An autoradiogram is
shown.
The open reading frame in Fig. 1A reveals that S-AKAP84 comprises 593 amino acids and has a
calculated M of 62,912. The amino acid composition
of S-AKAP84 is atypical; the protein is enriched in Glu, Pro, and Ser,
which account for 31% of the residues in the predicted sequence. The
abundance of Glu residues is reflected in the predicted isoelectric
point (pI = 4.55) and net charge(-36) of the polypeptide
at pH 7.4.
The sequence of S-AKAP84 is not homologous with protein
sequences compiled in the SWISS-PROT, GenBank, and PIR
data bases. However, the novel protein includes two short motifs that
may correspond to functional domains. Four tandem repeats of the
heptapeptide consensus sequence DRNEEGL are evident between amino acid
residues 310 and 338 (Fig. 1A). The beginning and end
of the reiterated region are closely bordered by the tripeptide NEE
(residues 306-308 and 341-343), which corresponds to the
central core of the consensus heptapeptide. The occurrence of a Leu
residue at the seventh position in each repeated unit raises the
possibility that this segment of S-AKAP84 constitutes a coiled-coil or
leucine zipper region(30) . Leucine zipper regions often
mediate the homo- and/or hetero-oligomerization of protein subunits. An
important caveat is that the heptapeptide repeats of S-AKAP84 lack an
internal hydrophobic residue that is conserved in prototypical leucine
zipper domains(30, 31) .
A lengthy hydrophobic
region (amino acid residues 8-26, Fig. 1A) is
evident near the NH terminus of S-AKAP84. The hydrophobic
domain and contiguous NH
- and COOH-terminal flanking
sequences were compared with classical ``signal sequences''
via computer programs (32, 33) . The results suggest
that this portion of S-AKAP84 does not constitute a classical signal
sequence capable of targeting the protein to a secretory pathway or the
plasma membrane. Instead, aspects of the organization and properties of
the NH
terminus of S-AKAP84 match more closely with
features described for the NH
-terminal segment of
NADH-cytochrome b
reductase(34, 35) . The key shared properties in
S-AKAP84 are as follows: (a) the first 7 residues include
basic and hydroxylated amino acids (Arg
, Ser
); (b) a long, extremely hydrophobic segment (19 residues)
provides a contiguous stop-transfer/hydrophobic anchor domain; (c) positively charged downstream residues (Arg
,
Lys
, Lys
, His
, His
; Fig. 1A) complete and stabilize the stop transfer
signal; and (d) acidic residues are excluded from the NH
terminus and first appear
10 residues downstream from the
end of the hydrophobic segment. Similar features target the NH
terminus of NADH-cytochrome b
reductase to
the outer membrane of mitochondria(35) . The stable,
post-translational insertion of the anchor sequence in the outer
membrane tethers this enzyme at a physiologically relevant location and
simultaneously ensures that its catalytic domain is exposed in the
cytoplasm. Locations and sequences of potentially important structural
motifs in the S-AKAP84 polypeptide are shown in Fig. 1B.
Figure 2: Characterization of antibodies directed against S-AKAP84. Western blots were prepared as described under ``Experimental Procedures.'' A, lanes 1 and 2 were loaded with 30 ng of purified P21 fusion protein; B, lanes 1 and 2 received 30 µg of particulate proteins from mouse testis. The membranes were probed with affinity-purified anti-S-AKAP84 IgGs (1:1000) as described under ``Experimental Procedures.'' Purified P21 (1 µg/ml) was included in the buffer when membranes containing the proteins fractionated in lane 2, (A) and lane 2 (B) were incubated with affinity-purified IgGs. The immunoblots were developed by the enhanced chemiluminescence procedure, and the signals were recorded on x-ray film. The weakly immunoreactive proteins (120 kDa and <84 kDa in testis (B, lane 1) are more evident in Fig. 7B, lane 5 (see below), where twice as much protein was analyzed and the time of exposure to x-ray film was increased by a factor of 2.5.
Figure 7:
Expression and RII binding activity of
S-AKAP84 in fractionated germ cells and testis. Samples of total
protein (30 µg) from pachytene spermatocytes (lane 1),
round spermatids (lane 2), condensing spermatids (lane
3), and mature sperm (lane 4) and an aliquot of the
particulate fraction (60 µg) of an adult testis homogenate (lane 5) were subjected to electrophoresis in a 0.1% SDS, 8.5%
polyacrylamide gel (see ``Experimental Procedures''). After
transfer to Immobilon P membranes, the size-fractionated proteins were
probed with P-labeled RII
(A) or
affinity-purified antibodies directed against S-AKAP84 (A) as
indicated under ``Experimental Procedures.'' The
chemiluminescence signals in B are proportional to the amount
of binding protein expressed; the autoradiogram in A shows the
RII
binding activity. Only the relevant portions of the gels are
shown. S-AKAP84 was not present in testis cytosol. When a replicate
Western blot was probed with antibodies that were preincubated with
excess recombinant S-AKAP84 or P21, no signals were
obtained.
Partial S-AKAP84 proteins were generated by
NH- and/or COOH-terminal truncation as indicated in Table 1. Signals indicating expression levels and RII
binding activities for a representative subset of mutant proteins are
shown in Fig. 3. Deletions of
200 amino acids from the COOH
and NH
termini of S-AKAP84 did not markedly alter specific
RII
binding activity (i.e. binding of
P-labeled ligand/mol of expressed protein) (Fig. 3A, lanes 1-7). However, when
residues 358-389 were eliminated, binding activity was
extinguished (Fig. 3A, lane 8). The absence of
RII
binding activity in a partial S-AKAP84 protein that contains
residues 358-593 (Fig. 3A, lane 9)
suggested that Glu
and adjacent NH
-terminal
residues (Fig. 1A) constitute a portion of the RII
tethering domain. Inspection of residues 355-376 in the S-AKAP84
sequence revealed that the positions of branched aliphatic side chains
(VIX
VLX
VX
AX
VC)
can be aligned in register with bulky aliphatic residues that mediate
the high affinity binding of RII
by neuronal AKAP75
(LLX
LVX
IX
IX
LV) (15) . The RII
tethering site in AKAP75 comprises amino
acids 392-413 and functions as an independent
domain(15) . Only one potentially significant difference is
noted in the comparison. The occurrence of Ala
in
S-AKAP84 conserves the hydrophobic character and
-helix-forming
propensity of Ile
in AKAP75, but the methyl side chain
contributes a smaller apolar surface. Substitution of branched chain
amino acids with Ala diminishes the avidity of AKAP75 for RII
.
However, the presence of neighboring Val
in S-AKAP84 may
compensate for this deficiency.
Figure 3:
Expression and RII binding activity
of truncated S-AKAP84 polypeptides. Deletion mutants of S-AKAP84 were
expressed in E. coli BL21 as described under
``Experimental Procedures.'' Samples (0.5 µg of total
protein; 20-50 ng of recombinant protein) of the soluble fraction
of bacterial lysates were subjected to electrophoresis in a 0.1% SDS,
10% polyacrylamide gel. After transfer to Immobilon P membranes, the
size-fractionated proteins were probed with
P-labeled
RII
(A) or antibodies directed against S-AKAP84 (B) as described under ``Experimental Procedures.''
The densitometric quantification of wild type and mutant S-AKAP84
proteins by antibody binding was used to normalize for variations in
the levels of recombinant protein expression. The autoradiogram in panel A shows RII
binding activities; the
chemiluminescence signals in panel B are proportional to the
amounts of the binding proteins. The nomenclature for recombinant wild
type and mutant RII
binding proteins is given in Table 1. Lanes 1-9 received extracts from E. coli expressing the
0,
N115,
N190,
N190-
C97,
N190-
C151,
N190-
C177,
N190-
C204,
N190-
C235, and
N358 proteins, respectively. The
recombinant protein in lane 9 was visualized by staining a
parallel lane loaded with 5 µg of total protein because the
epitopes bound by anti-S-AKAP84 IgGs were deleted. Calculated M
values for the various polypeptides are
indicated. Only the relevant regions of the autoradiograms are shown. E. coli extracts that contained the
N34 and
N190-
C124 proteins (see Table 1) yielded signals that
were similar to those observed for lanes 1-7;
N358-
C97 did not bind RII
(not
shown).
Based on the preceding
considerations a truncated S-AKAP84 protein that corresponds to amino
acids 331-443 was expressed in E. coli. Twenty-four
residues that precede the putative RII binding site were retained
because (a) this region is flanked by a long
NH-terminal segment in wild-type S-AKAP84 and (b)
there is no precedent for an RII binding domain that is congruent with
the NH
terminus of an AKAP (10, 11, 12, 13, 14, 15) .
The RII
binding activity of this mutant was similar to that
observed for S-AKAP84 and partial S-AKAP84 proteins in lanes
1-7 of Fig. 3A. Thus, amino acid residues
that govern RII binding activity in S-AKAP84 lie between residues 331
and 389.
Since Southern blots containing restriction enzyme digests of human genomic DNA yield a simple pattern that is consistent with a single-copy gene (data not shown), the Northern analysis suggests that S-AKAP84 RNA transcripts undergo alternative splicing. If multiple promoters drive transcription of the S-AKAP84 gene, they could also contribute to the diversity of transcripts. The smaller S-AKAP84 mRNA is expressed exclusively in testis (Fig. 4). PhosphorImager analysis disclosed that the testis-specific 3.2-kb mRNA is 10-100-fold more abundant than the 4.3-kb S-AKAP84 mRNA expressed in other tissues.
Figure 5: Expression of S-AKAP84 mRNA in the testes of mice of various ages. Total testis RNA was isolated from mice of the indicated ages as described by Carrera et al.(17) . The adult animals (A) were 10 weeks old. Samples of RNA (20 µg) were fractionated by electrophoresis under denaturing conditions and transferred to nitrocellulose as described under ``Experimental Procedures.'' The Northern blot was probed and calibrated as described in ``Experimental Procedures'' and Fig. 4. The size of the S-AKAP84 transcript is indicated. An autoradiogram is shown.
The expression pattern is
consistent with the appearance of S-AKAP84 mRNA during a late phase of
spermatid development. This possibility was addressed by monitoring the
expression of S-AKAP84 and its cognate mRNA in purified populations of
developing germ cells that correspond to meiotic pachytene
spermatocytes, postmeiotic round spermatids, condensing spermatids
(which undergo nuclear condensation and assume the elongated morphology
of mature sperm), and epididymal sperm. S-AKAP84 mRNA was not detected
in either pachytene spermatocytes or round spermatids (Fig. 6, lanes 2 and 3). In contrast, the 3.2-kb S-AKAP84
transcript is clearly evident in an enriched population of
condensing/elongating spermatids (Fig. 6, lane 4).
S-AKAP84 mRNA is not retained in mature sperm. The expression pattern
of an 84-kDa RII binding protein parallels the developmentally
controlled accumulation and disappearance of S-AKAP84 mRNA (Fig. 7A). A candidate 84-kDa anchor protein is also
independently identified by its avid binding with affinity-purified
anti-S-AKAP84 IgGs (Fig. 7B, lane 3). Moreover,
the binding assay and antibodies reveal an 84-kDa polypeptide that
corresponds to a major RII
binding protein in the particulate
fraction of testis homogenates (Fig. 7, A, lane
5, and B, lane 5). The highly sensitive
immunoblot-chemiluminescence assay (see above) reveals a low level of
the 84-kDa protein in round spermatids (Fig. 7B, lane 2). The lack of signals for S-AKAP84 mRNA (Fig. 6, lane 3) and RII binding activity (Fig. 7A, lane 2) in the round spermatid population may reflect lower
levels of sensitivity inherent in these measurements. It is also
possible that the 84-kDa protein in round spermatids fails to bind
RII
because its tethering function is modulated by developmental
stage-specific, post-translational modification. Overall, more than 90%
of S-AKAP84 immunoreactivity is associated with condensing/elongating
spermatids. (
)
Figure 6: Expression of S-AKAP84 mRNA in fractionated germ cells. Total RNA (20 µg/lane) from spleen (a negative control, lane 1), pachytene spermatocytes (lane 2), round spermatids (lane 3), and condensing spermatids (lane 4) were subjected to Northern gel analysis as described in the legend to Fig. 5and under ``Experimental Procedures.'' An autoradiogram is presented, and the size of S-AKAP84 mRNA is indicated.
The apparent M of the
murine RII
binding protein is estimated to be 84,000 when it is
compared with protein standards. Proteins of similar size are observed
when extracts of rat, human, and bovine testis are subjected to
RII
binding and immunoblot analyses.
Other tissues do
not contain the 84-kDa protein. The discrepancy between the calculated M
(63,000) and the apparent M
is probably due to the highly acidic and Pro-rich nature of the
protein (Fig. 1A). Proteins with these properties often
bind SDS poorly and assume atypical conformations under denaturing
conditions. Other AKAPs exhibit similar properties and have aberrantly
large apparent M
values(10, 26) .
A very low level of a 120-kDa protein is also evident in testis and
fractionated germ cells (Fig. 7B, lanes 2, 3, and 5). This protein (917 residues) is encoded by
a 3.1-kb, alternatively spliced S-AKAP84 mRNA. Structure/function analysis of the 120-kDa protein is in
progress.
Figure 8: S-AKAP84 is targeted selectively to the mitochondrial sheath. A representative example of a late condensing spermatid is presented. The phase contrast photograph (A) shows the characteristic morphology of the head containing the condensed nucleus, the midpiece with its characteristic ``corkscrew'' (helical) structure and the thinner principal piece. B, when the sample was probed with anti-S-AKAP84 IgGs and a fluorescein-conjugated secondary antibody, an intense fluorescence signal was obtained from the helical array of mitochondria in the midpiece. No comparable staining was observed for germ cells at any other stage of development. The signal observed in panel B was eliminated by preincubating the antibodies with an excess of the partial S-AKAP84 polypeptide antigen P21.
A cDNA encoding a novel, spermatid AKAP (S-AKAP84) has been
cloned and characterized. Previous investigations documented that
PKAII is associated with two distinct components of the
spermatozoan cytoskeleton: the fibrous sheath in the principal piece
and the outer membranes of an array of mitochondria located in the
midpiece(16) . Most of the RII
subunits are sequestered by
the mitochondria. Recently, Carrera et al.(17) demonstrated that an abundant cytoskeletal protein,
p82, mediates the anchoring of RII (PKAII) in the fibrous sheath.
Experiments described herein strongly implicate S-AKAP84 in the
delivery (targeting) of RII (PKAII) to the midpiece. The NH
terminus of S-AKAP84 has several structural features that
parallel the properties of signal/anchor domains (see
``Results''). Such domains target NADH-cytochrome b
reductase and several other proteins to the
outer mitochondrial membrane(35) . The properties of the
NH
-terminal segment of S-AKAP84 (residues 1-30) are
consistent with the location of the protein in situ.
Immunofluorescence microscopy demonstrated that S-AKAP84 is targeted
selectively to a large, helical structure in the midpiece of spermatids
undergoing nuclear condensation and tail elongation (Fig. 8).
The helical suprastructure corresponds to a precisely arranged assembly
of 70-80 mitochondria, otherwise known as the mitochondrial
sheath(36) . Thus, S-AKAP84 accumulates in a flagellar
compartment that also contains a high proportion of the anchored
RII
(PKII
) in mature
spermatozoa(3, 6, 16) . The highly
specialized germ cell mitochondria are components of the midpiece
cytoskeleton. In late postmeiotic stages of spermatogenesis, a portion
of the outer membrane of each mitochondrion closely apposes the outer
dense fibers (another cytoskeletal structure), whereas the remaining
mitochondrial surface directly contacts the cytoplasm.
The putative signal/anchor domain of S-AKAP84 is predicted to insert into the outer membrane bilayer post-translationally(35) . Thus, all other functional domains in organelle-sequestered S-AKAP84 molecules would be concentrated in a thin layer of cytoplasm adjacent to the mitochondrial surface. Therefore, the RII binding region located between residues 331 and 389 in S-AKAP84 would be accessible to mediate the tethering of cytoplasmic PKAII isoforms. PKAII captured by this mechanism could control or modulate flagellar activities by phosphorylating co-localized target substrates in the cytoskeleton, thereby altering their functional and/or structural properties.
The sequence of
S-AKAP84 diverges markedly from the primary structures of all other
AKAPs, with the exception of one short but potentially important
domain. A 22-residue segment of the RII binding region of S-AKAP84
(amino acids 355-376) contains a group of hydrophobic, branched
chain amino acids that align precisely with Ile, Leu, or Val residues
in the 22-amino acid sequence that constitutes the RII binding
domain of the neuronal AKAP, AKAP75(15) . Moreover, truncated
S-AKAP84 polypeptides that lack residues 355-358 or 359-376
have no RII binding activity (Fig. 3, and
``Results''). Since (a) the long, aliphatic side
chains in the tethering domain of AKAP75 are essential for high
affinity RII binding activity and (b) similar patterns of
hydrophobic, aliphatic side chains are evident in non-neuronal
AKAPs(10, 11) , it appears that the sequence
encompassed by residues 355-376 in S-AKAP84 may constitute a
tethering site. The inclusion of a 20-22-residue module that
conserves the spacing among long aliphatic side chains (and presumably
a precise orientation of hydrophobic surfaces in a higher order
structure) in otherwise divergent proteins, provides a mechanism for
targeting PKAII isoforms to a diverse array of intracellular target
sites (see Refs. 10 and 11).
Overall, the properties of S-AKAP84
fulfill criteria that are used in establishing the physiological
significance of an anchor protein. The polypeptide contains structural
features that appear to account for both the sequestration of RII and
the targeting/anchoring of AKAP-RII (or PKAII) complexes at a
previously established target site for cAMP action. Further
experiments, involving site-directed mutagenesis and
transfection/expression analysis, will be required to (a)
verify the proposed roles of the NH-terminal targeting and
centrally located tethering domains of S-AKAP84 and (b)
elucidate the exact roles of individual amino acids in each functional
domain.
The pattern of the developmental regulation of S-AKAP84
expression is quite atypical and provides additional, albeit indirect,
clues regarding the function of the anchor protein. The RII binding
protein and its cognate mRNA are apparently expressed de novo during late spermiogenesis (Fig. 5Fig. 6Fig. 7). Since the transcription of
most germ cell genes is silenced at or before this stage of development (37) , the data suggest that S-AKAP84 subserves a
``late'' developing, cAMP-mediated signaling pathway that
emerges with temporal precision. Furthermore, S-AKAP84 accumulation
occurs as the RII and RII
polypeptides reach their maximal
levels (1, 2) and in parallel with the remodeling and
reorganization of spermatid mitochondria(36) . In the latter
instance, classically shaped mitochondria are dispersed in the
cytoplasm of round spermatids; as the cells undergo nuclear
condensation and tail elongation the mitochondria migrate to the outer
dense fibers of the midpiece cytoskeleton and assume a crescent shape (36) . These mitochondria align end-to-end to form a helical
array. It is thought that preexisting mitochondria are modified by the
insertion of novel proteins during this period of transformation. Our
results indicate that S-AKAP84 and possibly S-AKAP84 complexed with
PKAII are incorporated into the outer mitochondrial membrane during
this major developmental transition. These observations raise the
possibility that S-AKAP84-PKAII complexes play a crucial role in
initiating or modulating this key step in sperm development.
Ultimately, it will be necessary to disrupt the S-AKAP84 gene or
otherwise compromise S-AKAP84 expression to establish the exact
physiological role of the anchor protein.
A puzzling aspect of the
results is that S-AKAP84 is not detected in mature mouse sperm. In
contrast, the association of RII with the mitochondrial sheath has
been documented by electron microscopy in mature bovine
sperm(16) . Several possible explanations may account for this
apparent discrepancy. Murine S-AKAP84 may be labile and subject to
artifactual degradation during the preparation of murine spermatozoa.
Another possibility is that S-AKAP84 plays a central physiological role
only in condensing spermatids and its vestigial retention in mature
sperm is species-specific and not physiologically relevant.
Alternatively, S-AKAP84 may target and deliver PKAII to the outer
mitochondrial membrane, whereupon the kinase is exchanged to a second,
more stable mitochondrial protein that permanently anchors the enzyme.
A 105-kDa protein that might fulfill this role was detected (via a low
intensity signal) in epididymal sperm (Fig. 7A, lane 4). This protein does not contain epitopes recognized by
anti-S-AKAP84 IgGs (Fig. 7B, lane 4). The
intracellular location and significance of the low level RII binding
activity associated with the 105-kDa protein remain to be investigated.
Irrespective of whether S-AKAP84 functions only in condensing
spermatids or over a broader span of development, it represents the
first example of a developmentally regulated, mitochondrially targeted
RII anchor protein.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34074[GenBank].