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Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.

Le Bot N, Antony C, White J, Karsenti E, Vernos I - J. Cell Biol. (1998)

Bottom Line: A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus.The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain.Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Program, European Molecular Biological Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

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Newly synthesized GalNacT2–GFP and GalT–GFP proteins do not reach the Golgi apparatus in cells expressing Xklp3–ST.  (A) A6 cells were transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP. Transfected cells (asterisks) were detected by immunofluorescence and confocal series were taken. A projection of six sections is presented. In cotransfected cells, GalNacT2–GFP is  seen in aggregates in the cytoplasm and in the ER. In control cells, cotransfected with Xklp1-ST–myc and GalNacT2–GFP, the GFP  marker localized to the Golgi apparatus. Bar, 10 μm. (B) Quantification of the defect of Golgi–GFP markers localization. A6 cells were  transiently transfected with the different combinations indicated. Cells were scored as either having the GFP Golgi marker localized  normally to the Golgi, to the ER and Golgi, or to the ER and aggregates. Bars show the percentage of cells in each category and the error bars show the standard deviation (n = 3). (C) Cells transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP exhibit an abnormal lectin staining. GalNacT2-GFP–expressing cells are detected by the GFP tag fluorescence. Cells were double labeled with a rabbit anti-HA antibody (then visualized by an AMCA conjugated anti-rabbit antibody) and the fluorescent lectin HP. Single confocal  planes are presented. Left, note that two cells transfected only with GalNacT2–GFP (asterisks) show normal labeling of the Golgi by the  HP lectin. Cotransfected cells show GalNacT2–GFP either in ER and Golgi (left panel, arrow 1) or in ER and aggregates (left panel, arrow 2; right panel). ∫Bar, 10 μm.
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Figure 9: Newly synthesized GalNacT2–GFP and GalT–GFP proteins do not reach the Golgi apparatus in cells expressing Xklp3–ST. (A) A6 cells were transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP. Transfected cells (asterisks) were detected by immunofluorescence and confocal series were taken. A projection of six sections is presented. In cotransfected cells, GalNacT2–GFP is seen in aggregates in the cytoplasm and in the ER. In control cells, cotransfected with Xklp1-ST–myc and GalNacT2–GFP, the GFP marker localized to the Golgi apparatus. Bar, 10 μm. (B) Quantification of the defect of Golgi–GFP markers localization. A6 cells were transiently transfected with the different combinations indicated. Cells were scored as either having the GFP Golgi marker localized normally to the Golgi, to the ER and Golgi, or to the ER and aggregates. Bars show the percentage of cells in each category and the error bars show the standard deviation (n = 3). (C) Cells transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP exhibit an abnormal lectin staining. GalNacT2-GFP–expressing cells are detected by the GFP tag fluorescence. Cells were double labeled with a rabbit anti-HA antibody (then visualized by an AMCA conjugated anti-rabbit antibody) and the fluorescent lectin HP. Single confocal planes are presented. Left, note that two cells transfected only with GalNacT2–GFP (asterisks) show normal labeling of the Golgi by the HP lectin. Cotransfected cells show GalNacT2–GFP either in ER and Golgi (left panel, arrow 1) or in ER and aggregates (left panel, arrow 2; right panel). ∫Bar, 10 μm.

Mentions: To examine whether the mutant form of Xklp3 blocked transport between the ER and the Golgi, we cotransfected cells with HA–Xklp3-ST and GalNacT2–GFP or GalT– GFP. The GFP markers localized exclusively to the Golgi in more than 80% of control cells. Cells expressing both HA–Xklp3-ST and one GFP Golgi marker showed a strong defect in the localization of the GFP marker to the Golgi (Fig. 9, A–C). Interestingly, the mislocalization of the Golgi GFP marker following co-transfection with the Xklp3-ST was associated with an aberrant HP lectin staining (Fig. 9 C). The phenotype observed could not be due to a general effect on the MTs because the cells still had a normal MT network focused at the MTOC (data not shown). As a control, we repeated the co-transfection experiment using a plasmid expressing a fragment of Xklp1 lacking the motor domain (Xklp1-ST–myc) similar to HA–Xklp3-ST. The Xklp1-ST–myc lacks also the NLS, and as a consequence remains in the cytoplasm, as HA– Xklp3-ST does. In cells expressing both Xklp1-ST–myc and GalNacT2–GFP, the GFP marker localized properly to the Golgi apparatus (Fig. 9 A).


Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.

Le Bot N, Antony C, White J, Karsenti E, Vernos I - J. Cell Biol. (1998)

Newly synthesized GalNacT2–GFP and GalT–GFP proteins do not reach the Golgi apparatus in cells expressing Xklp3–ST.  (A) A6 cells were transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP. Transfected cells (asterisks) were detected by immunofluorescence and confocal series were taken. A projection of six sections is presented. In cotransfected cells, GalNacT2–GFP is  seen in aggregates in the cytoplasm and in the ER. In control cells, cotransfected with Xklp1-ST–myc and GalNacT2–GFP, the GFP  marker localized to the Golgi apparatus. Bar, 10 μm. (B) Quantification of the defect of Golgi–GFP markers localization. A6 cells were  transiently transfected with the different combinations indicated. Cells were scored as either having the GFP Golgi marker localized  normally to the Golgi, to the ER and Golgi, or to the ER and aggregates. Bars show the percentage of cells in each category and the error bars show the standard deviation (n = 3). (C) Cells transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP exhibit an abnormal lectin staining. GalNacT2-GFP–expressing cells are detected by the GFP tag fluorescence. Cells were double labeled with a rabbit anti-HA antibody (then visualized by an AMCA conjugated anti-rabbit antibody) and the fluorescent lectin HP. Single confocal  planes are presented. Left, note that two cells transfected only with GalNacT2–GFP (asterisks) show normal labeling of the Golgi by the  HP lectin. Cotransfected cells show GalNacT2–GFP either in ER and Golgi (left panel, arrow 1) or in ER and aggregates (left panel, arrow 2; right panel). ∫Bar, 10 μm.
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Figure 9: Newly synthesized GalNacT2–GFP and GalT–GFP proteins do not reach the Golgi apparatus in cells expressing Xklp3–ST. (A) A6 cells were transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP. Transfected cells (asterisks) were detected by immunofluorescence and confocal series were taken. A projection of six sections is presented. In cotransfected cells, GalNacT2–GFP is seen in aggregates in the cytoplasm and in the ER. In control cells, cotransfected with Xklp1-ST–myc and GalNacT2–GFP, the GFP marker localized to the Golgi apparatus. Bar, 10 μm. (B) Quantification of the defect of Golgi–GFP markers localization. A6 cells were transiently transfected with the different combinations indicated. Cells were scored as either having the GFP Golgi marker localized normally to the Golgi, to the ER and Golgi, or to the ER and aggregates. Bars show the percentage of cells in each category and the error bars show the standard deviation (n = 3). (C) Cells transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP exhibit an abnormal lectin staining. GalNacT2-GFP–expressing cells are detected by the GFP tag fluorescence. Cells were double labeled with a rabbit anti-HA antibody (then visualized by an AMCA conjugated anti-rabbit antibody) and the fluorescent lectin HP. Single confocal planes are presented. Left, note that two cells transfected only with GalNacT2–GFP (asterisks) show normal labeling of the Golgi by the HP lectin. Cotransfected cells show GalNacT2–GFP either in ER and Golgi (left panel, arrow 1) or in ER and aggregates (left panel, arrow 2; right panel). ∫Bar, 10 μm.
Mentions: To examine whether the mutant form of Xklp3 blocked transport between the ER and the Golgi, we cotransfected cells with HA–Xklp3-ST and GalNacT2–GFP or GalT– GFP. The GFP markers localized exclusively to the Golgi in more than 80% of control cells. Cells expressing both HA–Xklp3-ST and one GFP Golgi marker showed a strong defect in the localization of the GFP marker to the Golgi (Fig. 9, A–C). Interestingly, the mislocalization of the Golgi GFP marker following co-transfection with the Xklp3-ST was associated with an aberrant HP lectin staining (Fig. 9 C). The phenotype observed could not be due to a general effect on the MTs because the cells still had a normal MT network focused at the MTOC (data not shown). As a control, we repeated the co-transfection experiment using a plasmid expressing a fragment of Xklp1 lacking the motor domain (Xklp1-ST–myc) similar to HA–Xklp3-ST. The Xklp1-ST–myc lacks also the NLS, and as a consequence remains in the cytoplasm, as HA– Xklp3-ST does. In cells expressing both Xklp1-ST–myc and GalNacT2–GFP, the GFP marker localized properly to the Golgi apparatus (Fig. 9 A).

Bottom Line: A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus.The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain.Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Program, European Molecular Biological Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

Show MeSH
Related in: MedlinePlus