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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 colocalization with different Golgi markers. (A) Double immunofluorescence on XL177 cells with the anti– Xklp3-tail antibody and antibodies against  different markers for the Golgi apparatus,  as indicated. Xklp3 colocalizes partially  with the lectin HP and completely with the  KDEL-receptor. (B) A6 cells stably expressing GalT–GFP or GalNacT2–GFP  were fixed and processed for immunofluorescence with the anti–Xklp3-tail antibody. Xklp3 colocalizes with both markers. Red, Xklp3; green, corresponding  marker. Bars, 10 μm.
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Figure 3: Xklp3 colocalization with different Golgi markers. (A) Double immunofluorescence on XL177 cells with the anti– Xklp3-tail antibody and antibodies against different markers for the Golgi apparatus, as indicated. Xklp3 colocalizes partially with the lectin HP and completely with the KDEL-receptor. (B) A6 cells stably expressing GalT–GFP or GalNacT2–GFP were fixed and processed for immunofluorescence with the anti–Xklp3-tail antibody. Xklp3 colocalizes with both markers. Red, Xklp3; green, corresponding marker. Bars, 10 μm.

Mentions: To determine if the interphase network stained by the anti–Xklp3-tail antibody corresponded to the Golgi apparatus, we performed double immunofluorescence labeling on XL177 cells with the anti–Xklp3-tail antibody and different markers of Golgi membranes. Xklp3 staining colocalized extensively with the Helix pomatia (HP) lectin that binds N-acetyl-galactosamine residues generated at the first step of O-linked glycosylation (Pavelka and Ellinger, 1985; Roth, 1984), in the Golgi apparatus (Fig. 3 A). It colocalized perfectly with the KDEL-receptor (revealed by an anti– KDEL-receptor antibody, Fig. 3 A), an itinerant Golgi protein which recycles KDEL-containing ligands from the Golgi back to the ER (Lewis and Pelham, 1992). The KDEL-receptor is usually localized in the cis-Golgi and the intermediate compartment (Tang et al., 1993). We also observed a perfect colocalization of Xklp3 with β-COP a component of the COP I complex implicated in transport steps between the ER and the Golgi (data not shown).


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 colocalization with different Golgi markers. (A) Double immunofluorescence on XL177 cells with the anti– Xklp3-tail antibody and antibodies against  different markers for the Golgi apparatus,  as indicated. Xklp3 colocalizes partially  with the lectin HP and completely with the  KDEL-receptor. (B) A6 cells stably expressing GalT–GFP or GalNacT2–GFP  were fixed and processed for immunofluorescence with the anti–Xklp3-tail antibody. Xklp3 colocalizes with both markers. Red, Xklp3; green, corresponding  marker. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132969&req=5

Figure 3: Xklp3 colocalization with different Golgi markers. (A) Double immunofluorescence on XL177 cells with the anti– Xklp3-tail antibody and antibodies against different markers for the Golgi apparatus, as indicated. Xklp3 colocalizes partially with the lectin HP and completely with the KDEL-receptor. (B) A6 cells stably expressing GalT–GFP or GalNacT2–GFP were fixed and processed for immunofluorescence with the anti–Xklp3-tail antibody. Xklp3 colocalizes with both markers. Red, Xklp3; green, corresponding marker. Bars, 10 μm.
Mentions: To determine if the interphase network stained by the anti–Xklp3-tail antibody corresponded to the Golgi apparatus, we performed double immunofluorescence labeling on XL177 cells with the anti–Xklp3-tail antibody and different markers of Golgi membranes. Xklp3 staining colocalized extensively with the Helix pomatia (HP) lectin that binds N-acetyl-galactosamine residues generated at the first step of O-linked glycosylation (Pavelka and Ellinger, 1985; Roth, 1984), in the Golgi apparatus (Fig. 3 A). It colocalized perfectly with the KDEL-receptor (revealed by an anti– KDEL-receptor antibody, Fig. 3 A), an itinerant Golgi protein which recycles KDEL-containing ligands from the Golgi back to the ER (Lewis and Pelham, 1992). The KDEL-receptor is usually localized in the cis-Golgi and the intermediate compartment (Tang et al., 1993). We also observed a perfect colocalization of Xklp3 with β-COP a component of the COP I complex implicated in transport steps between the ER and the Golgi (data not shown).

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