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A small surface hydrophobic stripe in the coiled-coil domain of type I keratins mediates tetramer stability.

Bernot KM, Lee CH, Coulombe PA - J. Cell Biol. (2005)

Bottom Line: Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers.The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo.The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Intermediate filaments (IFs) are fibrous polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus in higher eukaryotes. The mechanisms involved in bringing together approximately 16 elongated coiled-coil dimers to form an IF are poorly defined. Available evidence suggests that tetramer subunits play a key role during IF assembly and regulation. Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers. The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo. The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

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Keratin 16 protein turns over faster in K6  keratinocytes than in wild-type keratinocytes. (a) Primary keratinocytes were isolated from K6 or K17 wild-type and  mice. Equal amounts of protein extract were probed for K16 protein and β-tubulin (not depicted) as a loading control. K16 steady-state protein levels are greatly reduced in K6  keratinocytes compared with wild-type keratinocytes; whereas K16 steady-state protein levels increase in K17  keratinocytes. (b) Primary keratinocytes were isolated from K6 wild-type and  mice and pulse-chased with [35S]Met/Cys. Equal amounts of cell lysates were immunoprecipitated with antibodies against K16, K17, or K14. Immunoprecipitation was repeated two times to ensure maximal extraction of both labeled and unlabeled protein. Equal amounts of immunoprecipitated samples were separated via SDS-PAGE and autoradiographed. Complexes of keratins were pulled down in the immunoprecipitation; keratins are identified at the left, whereas the immunoprecipitating antibody is indicated above each autoradiograph. Autoradiographs were quantitated by densitometry. Bar graphs to the right show the percentage of label remaining at the various time points. K6αβ +/+ (black box) and K6αβ −/− (gray box).
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fig4: Keratin 16 protein turns over faster in K6 keratinocytes than in wild-type keratinocytes. (a) Primary keratinocytes were isolated from K6 or K17 wild-type and mice. Equal amounts of protein extract were probed for K16 protein and β-tubulin (not depicted) as a loading control. K16 steady-state protein levels are greatly reduced in K6 keratinocytes compared with wild-type keratinocytes; whereas K16 steady-state protein levels increase in K17 keratinocytes. (b) Primary keratinocytes were isolated from K6 wild-type and mice and pulse-chased with [35S]Met/Cys. Equal amounts of cell lysates were immunoprecipitated with antibodies against K16, K17, or K14. Immunoprecipitation was repeated two times to ensure maximal extraction of both labeled and unlabeled protein. Equal amounts of immunoprecipitated samples were separated via SDS-PAGE and autoradiographed. Complexes of keratins were pulled down in the immunoprecipitation; keratins are identified at the left, whereas the immunoprecipitating antibody is indicated above each autoradiograph. Autoradiographs were quantitated by densitometry. Bar graphs to the right show the percentage of label remaining at the various time points. K6αβ +/+ (black box) and K6αβ −/− (gray box).

Mentions: We previously showed that K16 could not compete effectively with K14 in the formation of stable heterotypic complexes in the presence of substoichiometric amounts of type II binding partners in vitro (K5 or K6; Paladini et al., 1996). The availability of suitable mouse models created an opportunity to monitor the fate of K16 protein under conditions of limited partner availability (K6 mice; Wong et al., 2000) or loss of a major type I keratin competitor (K17 mice; McGowan et al., 2002). Relative to wild-type control, the steady-state levels of K16 protein (but not mRNA) are much decreased in K6 skin keratinocytes in primary culture, in which K5 is the only type II keratin left and whose protein level does not become elevated (Fig. 4 a; Wong et al., 2000; Wong and Coulombe, 2003). In contrast, the levels of K14 and K17 proteins are not altered in these cells (Wong et al., 2000; Wong and Coulombe, 2003). Conversely, the levels of K16 protein, but not K14, are increased in K17 keratinocytes compared with control (Fig. 4 a). These data show that in a living cell context in which K5, K6α, K6β, K14, K15, K16, and K17 all coexist, K16 is uniquely sensitive to perturbations of the balance between types I and II keratins.


A small surface hydrophobic stripe in the coiled-coil domain of type I keratins mediates tetramer stability.

Bernot KM, Lee CH, Coulombe PA - J. Cell Biol. (2005)

Keratin 16 protein turns over faster in K6  keratinocytes than in wild-type keratinocytes. (a) Primary keratinocytes were isolated from K6 or K17 wild-type and  mice. Equal amounts of protein extract were probed for K16 protein and β-tubulin (not depicted) as a loading control. K16 steady-state protein levels are greatly reduced in K6  keratinocytes compared with wild-type keratinocytes; whereas K16 steady-state protein levels increase in K17  keratinocytes. (b) Primary keratinocytes were isolated from K6 wild-type and  mice and pulse-chased with [35S]Met/Cys. Equal amounts of cell lysates were immunoprecipitated with antibodies against K16, K17, or K14. Immunoprecipitation was repeated two times to ensure maximal extraction of both labeled and unlabeled protein. Equal amounts of immunoprecipitated samples were separated via SDS-PAGE and autoradiographed. Complexes of keratins were pulled down in the immunoprecipitation; keratins are identified at the left, whereas the immunoprecipitating antibody is indicated above each autoradiograph. Autoradiographs were quantitated by densitometry. Bar graphs to the right show the percentage of label remaining at the various time points. K6αβ +/+ (black box) and K6αβ −/− (gray box).
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fig4: Keratin 16 protein turns over faster in K6 keratinocytes than in wild-type keratinocytes. (a) Primary keratinocytes were isolated from K6 or K17 wild-type and mice. Equal amounts of protein extract were probed for K16 protein and β-tubulin (not depicted) as a loading control. K16 steady-state protein levels are greatly reduced in K6 keratinocytes compared with wild-type keratinocytes; whereas K16 steady-state protein levels increase in K17 keratinocytes. (b) Primary keratinocytes were isolated from K6 wild-type and mice and pulse-chased with [35S]Met/Cys. Equal amounts of cell lysates were immunoprecipitated with antibodies against K16, K17, or K14. Immunoprecipitation was repeated two times to ensure maximal extraction of both labeled and unlabeled protein. Equal amounts of immunoprecipitated samples were separated via SDS-PAGE and autoradiographed. Complexes of keratins were pulled down in the immunoprecipitation; keratins are identified at the left, whereas the immunoprecipitating antibody is indicated above each autoradiograph. Autoradiographs were quantitated by densitometry. Bar graphs to the right show the percentage of label remaining at the various time points. K6αβ +/+ (black box) and K6αβ −/− (gray box).
Mentions: We previously showed that K16 could not compete effectively with K14 in the formation of stable heterotypic complexes in the presence of substoichiometric amounts of type II binding partners in vitro (K5 or K6; Paladini et al., 1996). The availability of suitable mouse models created an opportunity to monitor the fate of K16 protein under conditions of limited partner availability (K6 mice; Wong et al., 2000) or loss of a major type I keratin competitor (K17 mice; McGowan et al., 2002). Relative to wild-type control, the steady-state levels of K16 protein (but not mRNA) are much decreased in K6 skin keratinocytes in primary culture, in which K5 is the only type II keratin left and whose protein level does not become elevated (Fig. 4 a; Wong et al., 2000; Wong and Coulombe, 2003). In contrast, the levels of K14 and K17 proteins are not altered in these cells (Wong et al., 2000; Wong and Coulombe, 2003). Conversely, the levels of K16 protein, but not K14, are increased in K17 keratinocytes compared with control (Fig. 4 a). These data show that in a living cell context in which K5, K6α, K6β, K14, K15, K16, and K17 all coexist, K16 is uniquely sensitive to perturbations of the balance between types I and II keratins.

Bottom Line: Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers.The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo.The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Intermediate filaments (IFs) are fibrous polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus in higher eukaryotes. The mechanisms involved in bringing together approximately 16 elongated coiled-coil dimers to form an IF are poorly defined. Available evidence suggests that tetramer subunits play a key role during IF assembly and regulation. Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers. The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo. The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

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