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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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Xklp3 does not colocalize with  ER the PDI or with post-Golgi structures  en route to the plasma membrane. (Top)  XL177 cells stained with the anti–Xklp3-tail antibody and an anti-KDEL signal antibody (anti-PDI). (Bottom) A6 cells transiently transfected with VSV-G–GFP and  stained with the anti–Xklp3-tail antibody.  Xklp3 colocalizes with VSV-G–GFP in  the Golgi apparatus but not in structures  at the vicinity of the plasma membrane,  thus indicating that Xklp3 is not associated with post-Golgi membranes. Red,  Xklp3; green, corresponding marker. Bar,  10 μm.
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Figure 4: Xklp3 does not colocalize with ER the PDI or with post-Golgi structures en route to the plasma membrane. (Top) XL177 cells stained with the anti–Xklp3-tail antibody and an anti-KDEL signal antibody (anti-PDI). (Bottom) A6 cells transiently transfected with VSV-G–GFP and stained with the anti–Xklp3-tail antibody. Xklp3 colocalizes with VSV-G–GFP in the Golgi apparatus but not in structures at the vicinity of the plasma membrane, thus indicating that Xklp3 is not associated with post-Golgi membranes. Red, Xklp3; green, corresponding marker. Bar, 10 μm.

Mentions: To determine whether Xklp3 was also present in the ER, we stained cells with the anti–Xklp3-tail antibody and an anti-PDI, generated against the KDEL motif of lumenal ER resident proteins (Tooze et al., 1989). Anti–Xklp3-tail and anti-PDI antibodies did not stain the same network of membranes (Fig. 4). To examine whether Xklp3 was present on ER-to-Golgi or TGN to plasma membrane vesicles, we transiently transfected A6 cells with a VSV-G–GFP. VSV-G–GFP is exported from the ER to the Golgi apparatus where it is processed before being included in post-TGN membrane-bound structures and transported towards the plasma membrane (Arnheiter et al., 1984; Kreis and Lodish, 1986). Xklp3 and VSV-G–GFP colocalized extensively in the Golgi region, but no colocalization was seen in the peripheral (i.e., ER or post-TGN) vesicle-like structures or at plasma membrane extensions.


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)

Xklp3 does not colocalize with  ER the PDI or with post-Golgi structures  en route to the plasma membrane. (Top)  XL177 cells stained with the anti–Xklp3-tail antibody and an anti-KDEL signal antibody (anti-PDI). (Bottom) A6 cells transiently transfected with VSV-G–GFP and  stained with the anti–Xklp3-tail antibody.  Xklp3 colocalizes with VSV-G–GFP in  the Golgi apparatus but not in structures  at the vicinity of the plasma membrane,  thus indicating that Xklp3 is not associated with post-Golgi membranes. Red,  Xklp3; green, corresponding marker. Bar,  10 μm.
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Related In: Results  -  Collection

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Figure 4: Xklp3 does not colocalize with ER the PDI or with post-Golgi structures en route to the plasma membrane. (Top) XL177 cells stained with the anti–Xklp3-tail antibody and an anti-KDEL signal antibody (anti-PDI). (Bottom) A6 cells transiently transfected with VSV-G–GFP and stained with the anti–Xklp3-tail antibody. Xklp3 colocalizes with VSV-G–GFP in the Golgi apparatus but not in structures at the vicinity of the plasma membrane, thus indicating that Xklp3 is not associated with post-Golgi membranes. Red, Xklp3; green, corresponding marker. Bar, 10 μm.
Mentions: To determine whether Xklp3 was also present in the ER, we stained cells with the anti–Xklp3-tail antibody and an anti-PDI, generated against the KDEL motif of lumenal ER resident proteins (Tooze et al., 1989). Anti–Xklp3-tail and anti-PDI antibodies did not stain the same network of membranes (Fig. 4). To examine whether Xklp3 was present on ER-to-Golgi or TGN to plasma membrane vesicles, we transiently transfected A6 cells with a VSV-G–GFP. VSV-G–GFP is exported from the ER to the Golgi apparatus where it is processed before being included in post-TGN membrane-bound structures and transported towards the plasma membrane (Arnheiter et al., 1984; Kreis and Lodish, 1986). Xklp3 and VSV-G–GFP colocalized extensively in the Golgi region, but no colocalization was seen in the peripheral (i.e., ER or post-TGN) vesicle-like structures or at plasma membrane extensions.

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