FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1970346586> ?p ?o ?g. }

• W1970346586 endingPage "34475" @default.
• W1970346586 startingPage "34467" @default.
• W1970346586 abstract "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. 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. type 1 hexokinase protein kinase A-anchoring protein green fluorescent protein germ cell-specific endoplasmic reticulum a unique amino-terminal domain encoded only by the HK1-sa transcript a unique internal domain encoded only by the HK1-sb transcript 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) (1Goldberg E. Methods Enzymol. 1975; 41: 318-323Crossref PubMed Scopus (28) Google Scholar, 2Storey B.T. Kayne F.J. Biol. Reprod. 1977; 16: 549-556PubMed Google Scholar). 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 (3Polakis P. G. Wilson J. E. Arch. Biochem. Biophys. 1985; 236: 328-337Crossref PubMed Scopus (109) Google Scholar,4Gelb B. Adams V. Jones S.N. Griffin L.D. MacGregor G.R. McCabe E.R.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 202-206Crossref PubMed Scopus (81) Google Scholar). 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 (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar). It is believed that the hydrophobic nature of this domain allows insertion of this region of the protein into the outer mitochondrial membrane (6Xie G.C. Wilson J.E. Arch. Biochem. Biophys. 1988; 267: 803-810Crossref PubMed Scopus (62) Google Scholar). 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 (7Murakami K. Piomelli S. Blood. 1997; 89: 762-766Crossref PubMed Google Scholar). 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 (8Katzen H.M. Adv. Enzyme Regul. 1967; 5: 335-356Crossref PubMed Scopus (135) Google Scholar, 9Katzen H.M. Soderman D.D. Cirillo V.J. Ann. N. Y. Acad. Sci. 1968; 151: 351-358Crossref PubMed Scopus (38) Google Scholar). 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 (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar, 11Mori C. Nakamura N. Welch J.E. Shiota K. Eddy E.M. Mol. Reprod. Dev. 1996; 44: 14-22Crossref PubMed Scopus (21) Google Scholar). Rather, the murine germ cell-specific HK mRNAs all encode an alternative 24-amino acid domain (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar), 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 (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar), only one, 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). 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 (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar). 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 (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar). 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 (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar, 13Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar, 14Visconti P.E. Olds-Clarke P. Moss S.B. Kalab P. Travis A.J. de las Heras M. Kopf G.S. Mol. Reprod. Dev. 1996; 43: 82-93Crossref PubMed Scopus (43) Google Scholar). 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 (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar). 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 (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar). 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 (15Faik P. Morgan M. Naftalin R.J. Rist R.J. Biochem. J. 1989; 260: 153-155Crossref PubMed Scopus (16) Google Scholar). 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 (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar). This processing might offer an explanation as to why HK1-sa transcript was not found at the protein level in a previous study (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar) and may represent a regulatory mechanism governing spatial and temporal expression of this protein during spermatogenesis. The pEGFP-N1 vector (CLONTECH, Palo Alto, CA) was utilized for the production of HK fusion proteins with a carboxyl-terminal GFP tag. This vector permitted the expression of the fusion proteins under the control of the cytomegalovirus promoter. Plasmids were transfected into M+R42 cells, a HK-deficient Chinese hamster ovary cell line (15Faik P. Morgan M. Naftalin R.J. Rist R.J. Biochem. J. 1989; 260: 153-155Crossref PubMed Scopus (16) Google Scholar). The M+R42 cell line was generously provided by Drs. Michael Morgan and Pelin Faik (Guy's Hospital Medical School, London, UK). Rhodamine 123 and DioC6(3) were purchased from Molecular Probes, Inc. (Eugene, OR). The cDNA clones encoding the different germ cell-specific HK1 isoforms were generously provided by E. M. Eddy (National Institutes of Environmental Health Sciences, Research Triangle Park, NC) (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar). Oligonucleotide primers for PCR were based upon the published sequences from Mori et al. (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar), and are listed in Table I. All constructs not containing mutations were generated by the same general method that is described below. A schematic representation of the constructs generated for use in this study is shown in Fig.1. To aid in the interpretation of the data, constructs have been named according to the domains that they possess.Table IOligonucleotide primers used for PCR in the generation of HK-GFP fusion proteinsConstructSense oligonucleotide primer1-aNucleotides are numbered according to the sequence for HK1-sa in Mori et al. (10).,1-bSense oligonucleotides contained a HindIII site at their 5′ end.Anti-sense oligonucleotide primer1-aNucleotides are numbered according to the sequence for HK1-sa in Mori et al. (10).,1-cAnti-sense oligonucleotides contained an EcoRI site at their 5′ end.tH275–2961-dA G at position 287 was converted to a C to remove an out-of-frame ATG.1616–1640GCS/tH78–1021616–1640GCS78–102272–296GCS/H78–1022572–2591tSA/GCS/tH47–671616–1640SA/GCS/tH2–251616–1640tSA/GCS47–67272–296SA/GCS2–25272–296tSA47–67172–193SA2–25172–193tH/SB275–2961616–1640GCS/tH/SB173–1901616–16401-a Nucleotides are numbered according to the sequence for HK1-sa in Mori et al. (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar).1-b Sense oligonucleotides contained a HindIII site at their 5′ end.1-c Anti-sense oligonucleotides contained an EcoRI site at their 5′ end.1-d A G at position 287 was converted to a C to remove an out-of-frame ATG. Open table in a new tab Briefly, constructs including the SA domain, or the truncated SA domain (tSA), were first amplified from mixed germ cell total RNA by reverse transcription PCR, using SuperScript II according to the manufacturer's instructions (Life Technologies, Inc.). For other constructs, the appropriate DNA regions were amplified with oligonucleotide primers using either the original cDNA clones or plasmids constructed previously as the template (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar). EitherTaq or Taq+ DNA polymerase was used depending upon the length of the expected product, according to the manufacturer's instructions (Stratagene, La Jolla, CA). All PCR products were visualized on 1% agarose gels with ethidium bromide and then isolated using the Wizard PCR Preps Purification Kit according to manufacturer's instructions (Promega, Madison, WI). The PCR products and the pEGFP-N1 vector were digested with HindIII andEcoRI (Promega, Madison, WI, and Life Technologies, Inc., respectively). The plasmid was dephosphorylated with calf intestinal alkaline phosphatase, and the PCR products were ligated into the plasmid with T4 DNA ligase (Life Technologies, Inc.). Plasmids then were transformed into HB101 competent cells (Promega), purified, and analyzed to check for the presence of the desired insert. DNA from plasmids containing inserts were sequenced on each end and over any internal domains of interest to ensure that the insert was correct and in-frame with the carboxyl-terminal GFP. An Applied Biosystems (Foster City, CA) model 373A automated sequencer, with the BigDye Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase FS, was utilized for sequencing. Transformed cells containing appropriate inserts were grown up, and plasmids were purified using the Perfect Prep Plasmid DNA kit (5 Prime → 3 Prime, Inc., Boulder, CO), prior to transfection into the M+R42 cells. Point mutations in the GCS domain were created using the Quik-Change Mutagenesis kit (Stratagene, La Jolla, CA). Amino acids were altered with the goals of changing relative charge or hydrophobicity, while keeping size relatively constant. Oligonucleotide primers (TableII) were constructed to conform to the guidelines of the kit and included single or multiple base changes that resulted in the desired expression of alternative amino acid residues. Mutant constructs were generated according to the instructions of the kit, with the exceptions that the annealing temperature was varied from 55 to 59 °C and the number of cycles was varied from 10–18, depending upon the construct. Briefly, sense and antisense primers covering the same region were generated and used for PCR off the circular plasmid. After PCR, the DNA was treated with DpnI for 1 h at 37 °C to digest the dam methylated parental plasmid. The mutated plasmid was then transformed into competent cells, isolated, and sequenced as above.Table IIOligonucleotide primers and specific nucleotide substitutions used in the generation of mutant HK-GFP fusion proteinsMutant construct2-aAmino acid residues are numbered from the first M in the GCS domain, from Mori et al. (10).Nucleotide substitutions2-bNucleotides are numbered according to the sequence for HK1-sa in Mori et al. (10).Oligonucleotide primers2-bNucleotides are numbered according to the sequence for HK1-sa in Mori et al. (10).mGCS(K16N)/tH241:A/C225–260mGCS(R18Q)/tH246:G/A225–260mGCS(K16N,R18Q)/tH241:A/C; 246:G/A225–260mGCS(P15A)/tH236:C/G224–268mGCS(P19A,P20A)/tH248:C/G; 251:C/G224–268mGCS(L21P,T22A,E23Q)/tH255:T/C; 257:A/G; 260:G/C245–272mGCS(del1–14)/tHNot applicablesense 236–2562-cA 5′ ATG was added to the sense oligonucleotide primer to begin translation at residue 15.,2-dSense oligonucleotides contained a HindIII site at their 5′ end.; anti-sense 1616–16402-eAnti-sense oligonucleotides contained an EcoRI site at their 5′ end.mGCS(P15A,K16N,R18Q,L21P,T22A,E23Q)tH236:C/G; 241:A/C; 246:G/A; 255:T/C; 257:A/G; 260:G/C225–2602-a Amino acid residues are numbered from the first M in the GCS domain, from Mori et al. (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar).2-b Nucleotides are numbered according to the sequence for HK1-sa in Mori et al. (10Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (78) Google Scholar).2-c A 5′ ATG was added to the sense oligonucleotide primer to begin translation at residue 15.2-d Sense oligonucleotides contained a HindIII site at their 5′ end.2-e Anti-sense oligonucleotides contained an EcoRI site at their 5′ end. Open table in a new tab Mutant constructs containing amino-terminal deletions were generated with oligonucleotide primers containing an in-frame ATG 5′ to the region where translation was to begin. The protocol for generating the construct was the same as described above for nonmutant constructs. For observation by confocal microscopy, M+R42 cells were cultured in chambered coverglasses (Lab-Tek catalog number 178655, Nalge Nunc International, Naperville, IL) and transfected using previously described procedures (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar). LipofectAMINE (Life Technologies, Inc.) was used as the transfection reagent. For preparation of cell extracts used in the immunoblotting experiments, M+R42 cells were grown in 100-mm plates. Transfection was done using LipofectAMINE PLUS to obtain higher transfection efficiencies, following the protocol supplied by the manufacturer. Extracts were prepared approximately 30 h after transfection. The cells were rinsed three times with phosphate-buffered saline, followed by the addition of 0.6 ml of hot (boiling water bath) sample buffer (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205416) Google Scholar). The cell lysates were transferred to microfuge tubes, sonicated for 6 s, and centrifuged for 5 min. The resultant supernatants were used for immunoblotting. As in the previous study (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar), fluorescence was observed using an Insight® confocal microscope (Meridian Instruments, Okemos, MI), generously made available by Dr. Melvin Schindler (Department of Biochemistry, Michigan State University). Excitation was obtained with the 488-nm line of an argon ion laser. Fluorescence of GFP was observed using a cutoff filter passing light of wavelength above 505 nm. Rhodamine 123 fluorescence was observed using a 570 ± 30 nm bandpass filter, and DioC6(3) fluorescence was observed with a 530 ± 30 nm bandpass filter. SDS-polyacrylamide gel electrophoresis on 6–20% gradient gels and electroblotting was performed as described previously (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar). Blots were blocked with 1% gelatin-5% nonfat dry milk in Tris-buffered saline and developed by standard methods, with detection using the SuperSignal West Pico Reagent (Pierce). Alternatively, proteins from cell lysates were separated and visualized using 10% gels (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar). Briefly, protein extracts from the transfected M+R42 cells were separated by SDS-polyacrylamide gel electrophoresis using 10% gels under reducing conditions (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205416) Google Scholar). Proteins were transferred from gels to Immobilon-P membranes (Millipore, Bedford, MA), which were then blocked in a Tris-buffered saline solution containing 0.1% Tween 20 (TTBS), and 5% cold water teleostean gelatin (Sigma). A variety of polyclonal antisera were used to detect the fusion protein products on immunoblots. Anti-GFP was purchased fromCLONTECH and used at a dilution of 1:10,000. An antiserum that detects both somatic and germ cell HK1 (anti-HK1) was used at a dilution of 1:10,000 (13Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar). Antisera against either the unique amino-terminal region of HK1-SA (anti-SA) or the germ cell-specific region shared by HK1-SA, HK1-SB, and HK1-SC (anti-GCS) were used at dilutions of 1:1,000 and 1:10,000, respectively (12Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (113) Google Scholar). Blots were probed with the appropriate primary antibody diluted in TTBS for 1 h, washed in TTBS, and then probed with an anti-rabbit, peroxidase-conjugated secondary antibody for 35 min. Blots were washed in TTBS for at least 2 h prior to visualization of target proteins by chemiluminescence (ECL, Amersham Pharmacia Biotech) and autoradiography. To examine the protein targeting of the germ cell-specific HK1 isoforms, we utilized an HK1-deficient somatic cell line, M+R42 cells, because no germ cell lines currently exist in culture that are permissive for spermatogenic differentiation. Confirming previous results (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar), sham-transfected cells (transfected with vector containing no insert) showed no fluorescence when viewed under conditions used to image GFP fluorescence in cells expressing GFP-constructs (results not shown). When stained with the mitochondrial marker, rhodamine 123, mitochondria were clearly visible in a tubular-punctate pattern in these cells (Fig. 2 A). Staining of cells with DioC6(3), which primarily stains the ER, revealed a reticular pattern in the perinuclear region (Fig.2 B). DioC6(3) also labeled some peri-nuclear mitochondria. However, the differences in the pattern of staining between the organelles and the comparison of this labeling to the rhodamine 123 pattern allowed the two organelles to be easily distinguished. When cells were transfected with a construct containing GFP alone, a diffuse fluorescence was observed throughout the cytoplasm and the nucleus that did not correlate with either ER or mitochondrial localization (Fig.2 C). In addition to these controls, it was necessary to ensure that domains within the HK sequence shared between the germ cell-specific and somatic isoforms did not play a role in protein targeting. It has been suggested that other regions within the amino-terminal half of HK1 might interact with the mitochondrial membrane-binding domain and confer additional targeting information (5Sui D. Wilson J.E. Arch. Biochem. Biophys. 1997; 345: 111-125Crossref PubMed Scopus (73) Google Scholar,17Smith A.D. Wilson J.E. Arch. Biochem. Biophys. 1991; 287: 359-366Crossref PubMed Scopus (27) Google Scholar). As a control for the possibility that these domains might confer specific targeting information, a construct containing the amino-terminal half of the germ cell-specific protein, minus the GCS region (tH) was generated. A diffuse pattern of fluorescence throughout the cytoplasm demonstrated that this construct did not contain sufficient intrinsic information to target fusion proteins in the absence of an amino-terminal targeting domain (Fig. 2 D). The amino-terminal GCS region is found in all three germ cell-specific HK1 mRNAs, where it replaces the mitochondrial membrane-binding domain of the somatic HK1 isoform. It is the only region of HK1-SC that differs from the somatic isoform and was therefore a likely candidate to be involved in protein targeting. A full-length construct based on HK1-SC, GCS/H, was generated and observed to localize to the ER, and to a lesser extent, the plasma membrane (Fig.3, A and B). A construct based on the amino-terminal half of HK1-SC including the GCS domain, GCS/tH, also was seen to target to the ER. However, in these cells, a larger proportion of signal was seen in the membranes of vesicles coming off the ER and incorporating into the plasma membrane (Fig. 3, C and D). To investigate more precisely the role of the GCS domain in ER targeting, the construct GCS, comprised solely of the GCS domain fused to GFP, was generated. This construct was targeted to the ER and vesicles coming off the ER, but the majority of the signal was found at the plasma membranes (Fig. 3,E and F). Comparison of localization patterns at different times following transfection with these constructs revealed a distinct trend that the smaller the construct, the faster and more efficient the processing to the plasma membrane (Fig. 3, A,C, and E, demonstrates such differences in the relative amounts of plasma membrane localization). Thus at comparable times after transfection, a major portion of the GCS was found in the plasma membrane, with a lesser amount of the GCS/tH, and only minor amounts of the GCS/H showing a plasma membrane localization. These data, taken together with those from the tH control, demonstrated that the GCS region was both necessary and sufficient for targeting GFP to the ER and plasma membranes in M+R42 cells. When imaging fields of cells transfected with the above constructs, there were several cells that showed a diffuse pattern of fluorescence typical for GFP alone. This phenomenon occurred at a higher frequency in cells" @default.
• W1970346586 created "2016-06-24" @default.
• W1970346586 creator A5024350807 @default.
• W1970346586 creator A5025662544 @default.
• W1970346586 creator A5030438643 @default.
• W1970346586 creator A5035905195 @default.
• W1970346586 creator A5044246398 @default.
• W1970346586 creator A5077520441 @default.
• W1970346586 creator A5086479392 @default.
• W1970346586 date "1999-11-01" @default.
• W1970346586 modified "2023-09-30" @default.
• W1970346586 title "A Novel NH2-terminal, Nonhydrophobic Motif Targets a Male Germ Cell-specific Hexokinase to the Endoplasmic Reticulum and Plasma Membrane" @default.
• W1970346586 cites W1528378220 @default.
• W1970346586 cites W1624097729 @default.
• W1970346586 cites W189961024 @default.
• W1970346586 cites W1985815362 @default.
• W1970346586 cites W1997021745 @default.
• W1970346586 cites W1999401753 @default.
• W1970346586 cites W2024879932 @default.
• W1970346586 cites W2028858498 @default.
• W1970346586 cites W2031704853 @default.
• W1970346586 cites W2032770293 @default.
• W1970346586 cites W2042781973 @default.
• W1970346586 cites W2048389722 @default.
• W1970346586 cites W2048994609 @default.
• W1970346586 cites W2067884529 @default.
• W1970346586 cites W2070361351 @default.
• W1970346586 cites W2088435636 @default.
• W1970346586 cites W2098488881 @default.
• W1970346586 cites W2100837269 @default.
• W1970346586 cites W2113031685 @default.
• W1970346586 cites W2139151974 @default.
• W1970346586 cites W2152869893 @default.
• W1970346586 cites W2157488384 @default.
• W1970346586 cites W2157502169 @default.
• W1970346586 cites W2161410278 @default.
• W1970346586 cites W2164948016 @default.
• W1970346586 cites W2197750417 @default.
• W1970346586 cites W2408822320 @default.
• W1970346586 cites W2419365590 @default.
• W1970346586 doi "https://doi.org/10.1074/jbc.274.48.34467" @default.
• W1970346586 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10567428" @default.
• W1970346586 hasPublicationYear "1999" @default.
• W1970346586 type Work @default.
• W1970346586 sameAs 1970346586 @default.
• W1970346586 citedByCount "30" @default.
• W1970346586 countsByYear W19703465862012 @default.
• W1970346586 countsByYear W19703465862013 @default.
• W1970346586 countsByYear W19703465862015 @default.
• W1970346586 countsByYear W19703465862018 @default.
• W1970346586 countsByYear W19703465862021 @default.
• W1970346586 countsByYear W19703465862023 @default.
• W1970346586 crossrefType "journal-article" @default.
• W1970346586 hasAuthorship W1970346586A5024350807 @default.
• W1970346586 hasAuthorship W1970346586A5025662544 @default.
• W1970346586 hasAuthorship W1970346586A5030438643 @default.
• W1970346586 hasAuthorship W1970346586A5035905195 @default.
• W1970346586 hasAuthorship W1970346586A5044246398 @default.
• W1970346586 hasAuthorship W1970346586A5077520441 @default.
• W1970346586 hasAuthorship W1970346586A5086479392 @default.
• W1970346586 hasConcept C104317684 @default.
• W1970346586 hasConcept C158617107 @default.
• W1970346586 hasConcept C162740809 @default.
• W1970346586 hasConcept C181199279 @default.
• W1970346586 hasConcept C185592680 @default.
• W1970346586 hasConcept C20251656 @default.
• W1970346586 hasConcept C2778106830 @default.
• W1970346586 hasConcept C2779122487 @default.
• W1970346586 hasConcept C2779826832 @default.
• W1970346586 hasConcept C41625074 @default.
• W1970346586 hasConcept C55493867 @default.
• W1970346586 hasConcept C86803240 @default.
• W1970346586 hasConcept C95444343 @default.
• W1970346586 hasConceptScore W1970346586C104317684 @default.
• W1970346586 hasConceptScore W1970346586C158617107 @default.
• W1970346586 hasConceptScore W1970346586C162740809 @default.
• W1970346586 hasConceptScore W1970346586C181199279 @default.
• W1970346586 hasConceptScore W1970346586C185592680 @default.
• W1970346586 hasConceptScore W1970346586C20251656 @default.
• W1970346586 hasConceptScore W1970346586C2778106830 @default.
• W1970346586 hasConceptScore W1970346586C2779122487 @default.
• W1970346586 hasConceptScore W1970346586C2779826832 @default.
• W1970346586 hasConceptScore W1970346586C41625074 @default.
• W1970346586 hasConceptScore W1970346586C55493867 @default.
• W1970346586 hasConceptScore W1970346586C86803240 @default.
• W1970346586 hasConceptScore W1970346586C95444343 @default.
• W1970346586 hasIssue "48" @default.
• W1970346586 hasLocation W19703465861 @default.
• W1970346586 hasOpenAccess W1970346586 @default.
• W1970346586 hasPrimaryLocation W19703465861 @default.
• W1970346586 hasRelatedWork W1181096670 @default.
• W1970346586 hasRelatedWork W1970346586 @default.
• W1970346586 hasRelatedWork W1982815320 @default.
• W1970346586 hasRelatedWork W2009793475 @default.
• W1970346586 hasRelatedWork W2020536777 @default.
• W1970346586 hasRelatedWork W2150351373 @default.
• W1970346586 hasRelatedWork W2158592428 @default.
• W1970346586 hasRelatedWork W2168143869 @default.

• next