Limits...
The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step.

Esk C, Chen CY, Johannes L, Brodsky FM - J. Cell Biol. (2010)

Bottom Line: Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells.CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer.In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome-TGN transport as critical for GLUT4 trafficking.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Clathrin heavy chain 22 (CHC22) is an isoform of the well-characterized CHC17 clathrin heavy chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome-TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport.

Show MeSH

Related in: MedlinePlus

Syntaxin 10 depletion partially phenocopies CHC22 depletion and is implicated in GLUT4 sequestration. (A–D) LHCNM2 human skeletal muscle myoblasts were differentiated, treated with control siRNA (A) or siRNA targeting STX10 (B and C) or CHC22 (D), and processed for immunofluorescence. Cells were double labeled using antibodies against STX10 (A, B, and D; green in merged insets), CHC22 (C; green in merged inset), and GLUT4 (red in merged insets) as indicated. Bars, 20 µm. (E–G) HeLa cells treated with control siRNA (E) or siRNA to deplete STX10 (F and G) levels were processed for immunofluorescence and labeled for STX10 (E and F; green in merged insets) and CI-MPR (E and F; red in merged insets) as indicated at the top. In G, fluorescent STxB (red in merged inset) in fresh medium was bound to cells for 30 min on ice, washed in PBS and chased for 60 min in fresh medium at 37°C, fixed, and processed for immunofluorescence using antibodies against GM130 (green in merged insets). (H) Detergent lysates of LHCNM2 cells treated with siRNA to deplete CHC17, CHC22, or STX10 or with control siRNA as indicated at the top were separated by SDS-PAGE and immunoblotted with antibodies against the proteins indicated at the left. α-Tubulin (α-tub) serves as a loading control. (I) Quantification of GLUT4 levels in detergent lysates of siRNA-treated cells generated as in H. Shown are levels ± SEM in the samples treated with the specific siRNA indicated under each bar compared with levels in cells treated with control siRNA in the same experiment (n = 6). P-values for selected samples are indicated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812854&req=5

fig10: Syntaxin 10 depletion partially phenocopies CHC22 depletion and is implicated in GLUT4 sequestration. (A–D) LHCNM2 human skeletal muscle myoblasts were differentiated, treated with control siRNA (A) or siRNA targeting STX10 (B and C) or CHC22 (D), and processed for immunofluorescence. Cells were double labeled using antibodies against STX10 (A, B, and D; green in merged insets), CHC22 (C; green in merged inset), and GLUT4 (red in merged insets) as indicated. Bars, 20 µm. (E–G) HeLa cells treated with control siRNA (E) or siRNA to deplete STX10 (F and G) levels were processed for immunofluorescence and labeled for STX10 (E and F; green in merged insets) and CI-MPR (E and F; red in merged insets) as indicated at the top. In G, fluorescent STxB (red in merged inset) in fresh medium was bound to cells for 30 min on ice, washed in PBS and chased for 60 min in fresh medium at 37°C, fixed, and processed for immunofluorescence using antibodies against GM130 (green in merged insets). (H) Detergent lysates of LHCNM2 cells treated with siRNA to deplete CHC17, CHC22, or STX10 or with control siRNA as indicated at the top were separated by SDS-PAGE and immunoblotted with antibodies against the proteins indicated at the left. α-Tubulin (α-tub) serves as a loading control. (I) Quantification of GLUT4 levels in detergent lysates of siRNA-treated cells generated as in H. Shown are levels ± SEM in the samples treated with the specific siRNA indicated under each bar compared with levels in cells treated with control siRNA in the same experiment (n = 6). P-values for selected samples are indicated.

Mentions: GLUT4 targeting to the GSC in human muscle depends on CHC22 (Vassilopoulos et al., 2009), and the studies reported here map CHC22 function in muscle to retrograde endosomal transport. In mice, which normally do not express CHC22, transgenically introduced CHC22 interacts with adaptors responsible for GLUT4 transport and disrupts murine GLUT4 traffic such that the formation and function of the GSC is impaired. It is conceivable that for CHC22 to function as it does in humans, additional species-specific factors are needed to complete its role in GSC formation (Vassilopoulos et al., 2009). An obvious candidate for such a factor is the SNARE family protein STX10, which plays a role in retrograde transport of CI-MPR from endosomes to the TGN, is enriched in muscle, and is expressed in humans but not mice (Tang et al., 1998; Ganley et al., 2008). The effects of STX10 depletion on GLUT4 traffic in human myoblasts were therefore assessed (Fig. 10) and found to phenocopy CHC22 depletion, causing dispersion and loss of GLUT4 staining (Fig. 10, B–D). As seen previously, STX10 depletion also caused dispersion of the CI-MPR in HeLa cells (Fig. 10 F), but it did not affect retrograde transport of STxB (Fig. 10 G; Mallard et al., 2002; Ganley et al., 2008). Again, phenocopying CHC22 (Vassilopoulos et al., 2009), STX10 depletion slightly reduced total GLUT4 levels in myotubes (Fig. 10, H and I), indicating that GLUT4 was partially degraded when GSC formation was disrupted. Localization of CHC22 was not severely affected in cells depleted of STX10 (Fig. 9 A, Fig. 10 C), whereas CHC22 depletion led to some dispersion of STX10 (Fig. 10, A and D). This suggests that CHC22 acts upstream of STX10 in endosomal sorting, consistent with CHC22 depletion affecting all three cargoes but STX10 depletion affecting only CI-MPR and GLUT4, but not STxB trafficking. Targeting STxB to the TGN must precede that of the other two cargoes, further subdividing retrograde transport from endosomes to TGN into two steps that follow a novel CHC22-dependent step. These data, which establish the joint participation of CHC22 and STX10 in GLUT4 traffic, map endosomal sorting and retrograde endosome-to-TGN transport as critical steps in GSC formation in human tissue.


The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step.

Esk C, Chen CY, Johannes L, Brodsky FM - J. Cell Biol. (2010)

Syntaxin 10 depletion partially phenocopies CHC22 depletion and is implicated in GLUT4 sequestration. (A–D) LHCNM2 human skeletal muscle myoblasts were differentiated, treated with control siRNA (A) or siRNA targeting STX10 (B and C) or CHC22 (D), and processed for immunofluorescence. Cells were double labeled using antibodies against STX10 (A, B, and D; green in merged insets), CHC22 (C; green in merged inset), and GLUT4 (red in merged insets) as indicated. Bars, 20 µm. (E–G) HeLa cells treated with control siRNA (E) or siRNA to deplete STX10 (F and G) levels were processed for immunofluorescence and labeled for STX10 (E and F; green in merged insets) and CI-MPR (E and F; red in merged insets) as indicated at the top. In G, fluorescent STxB (red in merged inset) in fresh medium was bound to cells for 30 min on ice, washed in PBS and chased for 60 min in fresh medium at 37°C, fixed, and processed for immunofluorescence using antibodies against GM130 (green in merged insets). (H) Detergent lysates of LHCNM2 cells treated with siRNA to deplete CHC17, CHC22, or STX10 or with control siRNA as indicated at the top were separated by SDS-PAGE and immunoblotted with antibodies against the proteins indicated at the left. α-Tubulin (α-tub) serves as a loading control. (I) Quantification of GLUT4 levels in detergent lysates of siRNA-treated cells generated as in H. Shown are levels ± SEM in the samples treated with the specific siRNA indicated under each bar compared with levels in cells treated with control siRNA in the same experiment (n = 6). P-values for selected samples are indicated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812854&req=5

fig10: Syntaxin 10 depletion partially phenocopies CHC22 depletion and is implicated in GLUT4 sequestration. (A–D) LHCNM2 human skeletal muscle myoblasts were differentiated, treated with control siRNA (A) or siRNA targeting STX10 (B and C) or CHC22 (D), and processed for immunofluorescence. Cells were double labeled using antibodies against STX10 (A, B, and D; green in merged insets), CHC22 (C; green in merged inset), and GLUT4 (red in merged insets) as indicated. Bars, 20 µm. (E–G) HeLa cells treated with control siRNA (E) or siRNA to deplete STX10 (F and G) levels were processed for immunofluorescence and labeled for STX10 (E and F; green in merged insets) and CI-MPR (E and F; red in merged insets) as indicated at the top. In G, fluorescent STxB (red in merged inset) in fresh medium was bound to cells for 30 min on ice, washed in PBS and chased for 60 min in fresh medium at 37°C, fixed, and processed for immunofluorescence using antibodies against GM130 (green in merged insets). (H) Detergent lysates of LHCNM2 cells treated with siRNA to deplete CHC17, CHC22, or STX10 or with control siRNA as indicated at the top were separated by SDS-PAGE and immunoblotted with antibodies against the proteins indicated at the left. α-Tubulin (α-tub) serves as a loading control. (I) Quantification of GLUT4 levels in detergent lysates of siRNA-treated cells generated as in H. Shown are levels ± SEM in the samples treated with the specific siRNA indicated under each bar compared with levels in cells treated with control siRNA in the same experiment (n = 6). P-values for selected samples are indicated.
Mentions: GLUT4 targeting to the GSC in human muscle depends on CHC22 (Vassilopoulos et al., 2009), and the studies reported here map CHC22 function in muscle to retrograde endosomal transport. In mice, which normally do not express CHC22, transgenically introduced CHC22 interacts with adaptors responsible for GLUT4 transport and disrupts murine GLUT4 traffic such that the formation and function of the GSC is impaired. It is conceivable that for CHC22 to function as it does in humans, additional species-specific factors are needed to complete its role in GSC formation (Vassilopoulos et al., 2009). An obvious candidate for such a factor is the SNARE family protein STX10, which plays a role in retrograde transport of CI-MPR from endosomes to the TGN, is enriched in muscle, and is expressed in humans but not mice (Tang et al., 1998; Ganley et al., 2008). The effects of STX10 depletion on GLUT4 traffic in human myoblasts were therefore assessed (Fig. 10) and found to phenocopy CHC22 depletion, causing dispersion and loss of GLUT4 staining (Fig. 10, B–D). As seen previously, STX10 depletion also caused dispersion of the CI-MPR in HeLa cells (Fig. 10 F), but it did not affect retrograde transport of STxB (Fig. 10 G; Mallard et al., 2002; Ganley et al., 2008). Again, phenocopying CHC22 (Vassilopoulos et al., 2009), STX10 depletion slightly reduced total GLUT4 levels in myotubes (Fig. 10, H and I), indicating that GLUT4 was partially degraded when GSC formation was disrupted. Localization of CHC22 was not severely affected in cells depleted of STX10 (Fig. 9 A, Fig. 10 C), whereas CHC22 depletion led to some dispersion of STX10 (Fig. 10, A and D). This suggests that CHC22 acts upstream of STX10 in endosomal sorting, consistent with CHC22 depletion affecting all three cargoes but STX10 depletion affecting only CI-MPR and GLUT4, but not STxB trafficking. Targeting STxB to the TGN must precede that of the other two cargoes, further subdividing retrograde transport from endosomes to TGN into two steps that follow a novel CHC22-dependent step. These data, which establish the joint participation of CHC22 and STX10 in GLUT4 traffic, map endosomal sorting and retrograde endosome-to-TGN transport as critical steps in GSC formation in human tissue.

Bottom Line: Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells.CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer.In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome-TGN transport as critical for GLUT4 trafficking.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Clathrin heavy chain 22 (CHC22) is an isoform of the well-characterized CHC17 clathrin heavy chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome-TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport.

Show MeSH
Related in: MedlinePlus