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A Novel NH2-terminal, Nonhydrophobic Motif Targets a Male Germ Cell-specific Hexokinase to the Endoplasmic Reticulum and Plasma Membrane*

Open AccessPublished:November 26, 1999DOI:https://doi.org/10.1074/jbc.274.48.34467
      Although three germ cell-specific transcripts of type 1 hexokinase exist in murine male germ cells, only one form, HK1-sc, is found at the protein level. This single isoform localizes to three distinct structures in mouse spermatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum (Travis, A. J., Foster, J. A., Rosenbaum, N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998) Mol. Biol. Cell 9, 263–276). The mechanism by which one protein is targeted to multiple sites within this highly polarized cell poses important questions of protein targeting. Because the study of protein targeting in germ cells is hampered by the lack of established cell lines in culture, constructs containing different domains of the germ cell-specific hexokinase transcripts were linked to a green fluorescent protein and transfected into hexokinase-deficient M+R42 cells. Constructs containing a nonhydrophobic, germ cell-specific domain, present at the amino terminus of the HK1-SC protein, were targeted to the endoplasmic reticulum and the plasma membrane. Mutational analysis of this domain demonstrated that a complex motif,PKIRPPLTE(with essential residues italicized), represented a novel endoplasmic reticulum-targeting motif. Constructs based on another germ cell-specific hexokinase transcript, HK1-sa, demonstrated the specific proteolytic removal of an amino-terminal domain, resulting in a protein product identical to HK1-SC. Such processing might constitute a regulatory mechanism governing the spatial and/or temporal expression of the protein.
      HK1
      type 1 hexokinase
      AKAP
      protein kinase A-anchoring protein
      GFP
      green fluorescent protein
      GCS
      germ cell-specific
      ER
      endoplasmic reticulum
      SA
      a unique amino-terminal domain encoded only by the HK1-sa transcript
      SB
      a unique internal domain encoded only by the HK1-sb transcript
      PCR
      polymerase chain reaction
      The targeting of proteins to specific organelles or biochemical compartments within a cell is critical for normal cellular function. The biological significance of appropriate protein targeting is best demonstrated in cells that are highly polar in organization. For example, epithelial cells of the renal tubules and intestinal lumen could not provide directional transport without their sodium-potassium ATPases organized strictly on their basolateral surfaces. Among the various cell types that have been studied, mature spermatozoa represent one of the most highly differentiated and polarized cells. The spermatozoon can be divided into three main compartments: the head, the midpiece, and the principal piece of the flagellum. Within these different compartments are many unique organelles such as the membrane-delimited acrosome in the head, as well as the fibrous sheath and outer dense fibers, cytoskeletal elements that surround the axoneme in the flagellum. Furthermore, organelles common to both germ cells and somatic cells possess unusual adaptations in the male gamete. In this regard, sperm mitochondria differ from their somatic counterparts in that they are restricted to a specific region of the cell (the midpiece of the flagellum) and possess additional germ cell-specific isozymes (e.g. lactate dehydrogenase-X) (
      • Goldberg E.
      ,
      • Storey B.T.
      • Kayne F.J.
      ). By regionalizing the distribution of specific organelles and proteins, spermatozoa have achieved a functional compartmentalization of the machinery necessary for such diverse functions as cellular motility, binding and penetrating the extracellular matrix of the egg, and binding and fusing with the plasma membrane of the egg. How these components are targeted and assembled during spermatogenesis to form the polarized spermatozoon is largely unknown.
      One enzyme critical to the compartmentalized metabolic pathways of both spermatozoa and somatic tissues is type 1 hexokinase (HK1)1 This enzyme is best known for catalyzing the phosphorylation of glucose in the first step of glycolysis. Targeting of the somatic isoforms of HK1 is based largely upon the presence of different amino-terminal domains. The classical somatic cell HK1 can associate with the outer mitochondrial membrane through a 15-amino acid amino-terminal hydrophobic domain (
      • Polakis P.
      • G.
      • Wilson J.
      • E.
      ,
      • Gelb B.
      • Adams V.
      • Jones S.N.
      • Griffin L.D.
      • MacGregor G.R.
      • McCabe E.R.B.
      ). This “mitochondrial membrane-binding domain” has been shown to be sufficient to target a green fluorescent protein (GFP) construct to the mitochondria in M+R42 cells, a HK-deficient cell line (
      • Sui D.
      • Wilson J.E.
      ). It is believed that the hydrophobic nature of this domain allows insertion of this region of the protein into the outer mitochondrial membrane (
      • Xie G.C.
      • Wilson J.E.
      ).
      Two cell types, reticulocytes and male germ cells, contain mRNAs encoding variants of HK1 that do not possess this mitochondrial membrane-binding domain. In human reticulocytes, one of these HK variants contains an alternative amino terminus that replaces the first 21 residues with 20 alternative amino acids (
      • Murakami K.
      • Piomelli S.
      ). Interestingly, this reticulocyte-specific HK isozyme does not target to mitochondria but rather is found exclusively in the cytosol.
      It has been known for some time that male germ cells possess a variant of HK1 protein (
      • Katzen H.M.
      ,
      • Katzen H.M.
      • Soderman D.D.
      • Cirillo V.J.
      ). In both mice and humans, three cDNAs have been identified that are predicted to encode for male germ cell-specific isoforms of HK1 that do not contain either the mitochondrial membrane-binding domain or the reticulocyte-specific sequence (
      • Mori C.
      • Welch J.E.
      • Fulcher K.D.
      • O'Brien D.A.
      • Eddy E.M.
      ,
      • Mori C.
      • Nakamura N.
      • Welch J.E.
      • Shiota K.
      • Eddy E.M.
      ). Rather, the murine germ cell-specific HK mRNAs all encode an alternative 24-amino acid domain (
      • Mori C.
      • Welch J.E.
      • Fulcher K.D.
      • O'Brien D.A.
      • Eddy E.M.
      ), called GCS for itsgerm cell-specific expression. This GCS domain is not hydrophobic in nature, in contrast to the somatic mitochondrial membrane-binding domain.
      Although three germ cell-specific HK1 transcripts, HK1-sa, HK1-sb, and HK1-sc, have been identified in the mouse (
      • Mori C.
      • Welch J.E.
      • Fulcher K.D.
      • O'Brien D.A.
      • Eddy E.M.
      ), only one, HK1-SC,
      Lowercase letters will be used to denote nucleic acids/transcripts (e.g. HK1-sc), and uppercase letters will be used to denote proteins (e.g. HK1-SC).
      2Lowercase letters will be used to denote nucleic acids/transcripts (e.g. HK1-sc), and uppercase letters will be used to denote proteins (e.g. HK1-SC).
      has been demonstrated to be expressed at the protein level (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ). Our interest in the targeting of proteins within male germ cells began with our demonstration that this isoform is found associated with the membranes of the head of the sperm, as well as with the mitochondria and the fibrous sheath of the flagellum (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ). How one isoform of HK could target to such dissimilar biochemical structures within a single cell type poses interesting questions regarding the basic cell biology of protein targeting and metabolic compartmentalization, as well as of sperm cell assembly during spermatogenesis.
      In addition to its unusual localization pattern, HK1-SC has some biochemical characteristics that are not seen with other isoforms of HK1. For example, the murine germ cell-specific protein is phosphorylated on tyrosine residues, and at least a population of this protein has properties consistent with an integral membrane protein (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ,
      • Kalab P.
      • Visconti P.
      • Leclerc P.
      • Kopf G.S.
      ,
      • Visconti P.E.
      • Olds-Clarke P.
      • Moss S.B.
      • Kalab P.
      • Travis A.J.
      • de las Heras M.
      • Kopf G.S.
      ). Both the localization of the germ cell-specific HK to cell membranes and its biochemical behavior as an integral membrane protein are unexpected, given the fact that HK1-SC lacks the mitochondrial membrane-binding domain and contains neither predicted signal sequences nor regions of significant hydrophobicity (
      • Mori C.
      • Welch J.E.
      • Fulcher K.D.
      • O'Brien D.A.
      • Eddy E.M.
      ).
      Studies of protein targeting in germ cells have been hampered by the lack of established germ cell lines in culture. Therefore, to initiate a study of the mechanism by which HK may be targeted to multiple subcellular locations in sperm, we have chosen a heterologous cell expression system previously found to be useful in studies of the targeting of somatic forms of mammalian HK (
      • Sui D.
      • Wilson J.E.
      ). A series of HK-GFP fusion constructs including different segments of the murine germ cell-specific isoforms, or mutants thereof, were expressed in the HK-deficient M+R42 cell line (
      • Faik P.
      • Morgan M.
      • Naftalin R.J.
      • Rist R.J.
      ). Subcellular distribution was monitored using confocal fluorescence microscopy. The nonhydrophobic GCS domain was found to be necessary and sufficient to target fusion proteins to the ER and plasma membrane in this expression system. Mutational analysis defined a specific and complex targeting motif located within the carboxyl-terminal 10 amino acids of the GCS domain. Individual point mutations and several combinations of mutations within this region did not abolish ER targeting but did disrupt normal protein processing through the ER to the plasma membrane. Only when six specific residues were mutated in combination was targeting to the ER abolished. Constructs based on another germ cell-specific HK1 transcript, HK1-sa, revealed the specific proteolytic removal of the unique amino-terminal domain of this isoform. The cleavage of this domain resulted in a protein identical to HK1-SC, the isoform of HK1 previously demonstrated to be expressed in sperm (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ). This processing might offer an explanation as to why HK1-sa transcript was not found at the protein level in a previous study (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ) and may represent a regulatory mechanism governing spatial and temporal expression of this protein during spermatogenesis.

      DISCUSSION

      In highly polarized cells, the correct targeting of proteins to defined structures or compartments is critical for localizing specific functions to limited regions of the cell. In spermatozoa, the head is the carrier of the paternal genetic material, as well as being the region of the cell responsible for interacting first with the zona pellucida, the extracellular matrix of the oocyte, and then with the plasma membrane of the oocyte. The flagellum is highly specialized to provide the sperm with progressive motility and is itself subdivided into different sections; the midpiece is the only region of the sperm that contains mitochondria, and both the midpiece and principal piece contain cytoskeletal scaffolding proteins that surround the axoneme. Our previous finding that one germ cell-specific isozyme of HK1 localizes to the membranes of the head, the mitochondria of the midpiece and the fibrous sheath of the flagellum (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ) raised the question of how one protein can be targeted to such distinct biochemical compartments within a single cell type.
      Because no lines of germ cells exist that are permissive for morphological differentiation into spermatozoa in culture, constructs containing different domains of the germ cell-specific HK1 transcripts HK1-sa, HK1-sb, and HK1-sc were linked to a carboxyl-terminal GFP marker and transfected into a somatic cell line deficient in HK. Although this system would not permit the study of targeting to the fibrous sheath (a structure that has no analogs in other cell types), it allows for the study of interactions with cell membranes, mitochondria, and other cytoskeletal components.
      We found that the addition of the GCS domain, when expressed in this somatic cell system, was both necessary and sufficient to target HK-GFP fusion proteins to the ER and the plasma membrane. The targeting of this fusion protein to the ER and plasma membranes is consistent with the localization of the endogenous protein to the membranes of the sperm head (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ). Moreover, the appearance of the fusion proteins in the ER and plasma membranes demonstrated that the targeting mechanism was not germ cell-specific and that this heterologous system possessed the necessary cellular machinery for ER localization and translocation to the plasma membrane.
      Targeting of the GCS domain to the ER and plasma membrane occurred despite the absence of a hydrophobic signal sequence. Precedent exists for plasma membrane or extracellular localization of proteins that are not hydrophobic in nature. For example, a muscle lectin has been shown to be “externalized” from cells by means of a novel secretory mechanism (
      • Cooper D.N.
      • Barondes S.H.
      ). However, the model suggested for this particular protein requires that it maintains a cytosolic localization in vesicles that are themselves released from the cell and then opened (
      • Cooper D.N.
      • Barondes S.H.
      ). In contrast, the endogenous HK1-SC protein is not soluble. Indeed, at least some of the protein has the biochemical characteristics of an integral membrane protein, in that it is not solubilized by conditions that dissociate most peripheral membrane proteins, nor is it solubilized by conditions that dissociate somatic HK from mitochondria (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ,
      • Visconti P.E.
      • Olds-Clarke P.
      • Moss S.B.
      • Kalab P.
      • Travis A.J.
      • de las Heras M.
      • Kopf G.S.
      ). The fusion proteins generated in this study appeared to be associated with the membranes of vesicles that integrated into the plasma membrane, so it is unlikely that they were externalized by the mechanism used by the muscle lectin.
      Most proteins are targeted to the ER by a signal recognition particle-mediated pathway that recognizes and binds a hydrophobic signal sequence on the amino terminus of a protein (for review see Ref.
      • Schatz G.
      • Dobberstein B.
      ). As noted, the GCS domain does not contain a hydrophobic region. Rather, the results of our mutational analysis demonstrated that a novel motif, PKIRPPLTE (with residues in bold print appearing to be of particular importance), was responsible for ER targeting. Whether this motif operates by being bound in the signal recognition particle-mediated pathway or is processed through a signal recognition particle-independent pathway is unknown. Similarly, whether membrane targeting takes place through a direct interaction of this motif with the membranes or secondarily through the interaction of this motif with another membrane protein will form the basis of future study.
      Mutations of individual amino acids within this novel motif led to a disruption of the ER, characterized by an increased “vesiculation” and loss of the normal reticular appearance. Based on the appearance of cells expressing these mutant constructs at various times after transfection, it seems that initial ER targeting was intact but that either the ER membranes became fragmented into vesicles or that processing through the Golgi lost directionality and normal vesicles dispersed throughout the cell. A similar result was seen when the 14 amino acids amino-terminal to the targeting motif were removed, and the strong conservation between the mouse and human sequences of the amino-terminal 7 residues within the GCS domain suggests an important role for those residues. These results suggest that those amino-terminal amino acids might also interact with the ER membranes in some way to influence processing to the Golgi and plasma membrane or that they might interact with other proteins that contribute to the normal ultrastructural morphology of the ER.
      The time course data with the mutant constructs also revealed that at later times after transfection the majority of the disrupted vesicles aggregated at the cell periphery underlying the plasma membrane. It has been reported that somatic HK can translocate to the cell cortex of activated rat macrophages (
      • Pedley K.C.
      • Jones G.E.
      • Magnani M.
      • Rist R.J.
      • Naftalin R.J.
      ), presumably through an interaction with cytoskeletal elements. Whether our fusion proteins congregated in the cell periphery because they were still able to interact with specific components of the plasma membrane or whether there were secondary interactions with cytoskeletal elements or some other translocating mechanism is unknown.
      When the GCS domain was linked directly to GFP or to the full-length HK sequence, proteolytic removal of the GCS domain occurred at higher levels than when the GCS domain was linked to a half-length HK sequence. One possible explanation for this trend is that the additional HK sequence provided limited stability against protease activity. Proteolytic degradation of amino-terminal domains was not limited to the GCS region. Fusion proteins based on the HK1-sa sequence, regardless of which initiator methionine was used, showed the specific removal of the SA domain. That this processing occurred in a heterologous system suggested that the protease was not germ cell-specific. The only clear instances of mitochondrial targeting observed in this study were seen with fusion proteins containing the SA domain. It is tempting to speculate that the proteolytic removal of the SA domain immediately amino-terminal to the GCS domain might regulate a chimeric targeting motif. Precedent for such chimeric targeting motifs that result in either ER or mitochondrial localization dependent upon motif processing exists (
      • Addya S.
      • Anandatheerthavarada H.K.
      • Biswas G.
      • Bhagwat S.V.
      • Mullick J.
      • Avadhani N.G.
      ). However, the susceptibility of fusion proteins containing the SA domain linked to GFP either directly (tSA and SA) or with an intervening GCS domain (tSA/GCS and SA/GCS) to endogenous proteolytic degradation precluded conclusions on this matter.
      Recently, the localization of a protein kinase A-anchoring protein (D-AKAP1) to either the mitochondria or to the ER was shown to result from the alternative splicing of different amino-terminal domains (
      • Huang L.J.
      • Wang L.
      • Ma Y.
      • Durick K.
      • Perkins G.
      • Deerinck T.J.
      • Ellisman M.H.
      • Taylor S.S.
      ). This finding is noteworthy in two ways. First, it provides an alternative method of targeting a given protein to two different organelles. Interestingly, the HK1-sa, HK1-sb, HK1-sc, and somatic HK1 transcripts also arise from alternative splicing of a single gene (
      • Mori C.
      • Nakamura N.
      • Welch J.E.
      • Gotoh H.
      • Goulding E.H.
      • Fujioka M.
      • Eddy E.M.
      ). Should the proteolytic processing of the SA domain seen in the present study be involved in the targeting of that isoform in germ cells, then the targeting of HK1 in germ cells would be regulated by alternative splicing as well as by the processing of targeting domains. This added complexity would also suggest that the GCS domain, which is included in both transcripts, might be necessary in protein function or at least to maintain an association with a given membrane, be it ER, plasma membrane, or outer mitochondrial membrane.
      The second interesting comparison with the D-AKAP1 findings relates to the nature of its mitochondrial membrane-binding domain. This domain is similar in amino acid sequence to the mitochondrial membrane-binding domain of the somatic HK1 (
      • Huang L.J.
      • Wang L.
      • Ma Y.
      • Durick K.
      • Perkins G.
      • Deerinck T.J.
      • Ellisman M.H.
      • Taylor S.S.
      ). It is intriguing that alternative splice variant domains responsible for the mitochondrial localization of two proteins that can target to multiple organelles have such similarity. Whether this similarity in mitochondrial targeting domains is the result of convergent evolution or a common ancestor for that domain is unknown. D-AKAP1 was originally characterized in spermatids as S-AKAP84, found in the mitochondria of the midpiece, and was suggested to localize to that organelle by means of an amino-terminal targeting motif (
      • Lin R.Y.
      • Moss S.B.
      • Rubin C.S.
      ). Other AKAPs localize to the fibrous sheath of the principal piece (
      • Carrera A.
      • Gerton G.L.
      • Moss S.B.
      ), and we have suggested that the co-localization of an AKAP and glycolytic enzymes in the fibrous sheath might allow ATP to be produced at the point where it is needed to be used in the regulation of sperm motility (
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      ). Given the critical nature of the flagellar ultrastructure in sperm function and the absence of translocating mechanisms in mature spermatozoa, the assembly of the flagellum and the inherent targeting of its components in germ cells takes on added importance.
      Along with differences in sequence, fusion protein targeting to the mitochondria seen with constructs tSA/GCS/tH and SA/GCS/tH might be dependent upon time of expression relative to the cell cycle. Endogenous HK1-sa is expressed in meiotic germ cells, whereas HK1-sc is expressed post-meiotically (
      • Mori C.
      • Welch J.E.
      • Fulcher K.D.
      • O'Brien D.A.
      • Eddy E.M.
      ,
      • Visconti P.E.
      • Olds-Clarke P.
      • Moss S.B.
      • Kalab P.
      • Travis A.J.
      • de las Heras M.
      • Kopf G.S.
      ). The possibility that only the subset of cells transfected at a specific stage of the cell cycle would show mitochondrial localization might explain why mitochondrial targeting was seen in such a limited number of cells. Alternatively, the fact that somatic mitochondria are substantially different from sperm mitochondria (), might lower the efficiency with which the germ cell-specific domains interact with the somatic mitochondria. Lastly, an example of a protein being targeted to the Golgi in a somatic tissue and in early stages of germ cell development, but to the midpiece of mature spermatozoa, also exists (
      • Saberan-Djoneidi D.
      • Picart R.
      • Escalier D.
      • Gelman M.
      • Barret A.
      • Tougard C.
      • Glowinski J.
      • Levi-Strauss M.
      ). This finding suggests that targeting to the ER can precede targeting to mitochondria in germ cells.
      Germ cells possess organelles such as the fibrous sheath, which is not found in somatic cells. Moreover, even organelles common to germ and somatic cells (e.g. mitochondria) differ in details of their structure and function. Thus it is evident that studies such as the present one, employing a heterologous system for expression of germ cell-specific proteins, has its limitations. Nonetheless, such studies provide a feasible approach for detecting sequence elements that may play a targeting role in germ cells. In future work, the significance of such elements in germ cell differentiation in vivo will be explored using transgenic animal models that allow expression in the appropriate cellular environment and temporal pattern.

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      1. ****

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