Limits...
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.

Show MeSH

Related in: MedlinePlus

Xklp3 and the KDEL  receptor have a similar behavior upon BFA treatment.  (A) A6 cells expressing Gal  NacT2–GFP constitutively  were incubated with 5 αg/ml  of BFA for 5 min or 1 h and  then stained with the anti– Xklp3-tail antibodies. After  5 min of BFA treatment,  GalNacT2–GFP starts to be  redistributed into the ER often appearing on tubular  structures. The anti–Xklp3-tail antibody gives a punctuate staining dispersed in the  cytoplasm, often associated  to but not overlapping with  the GalNacT2–GFP staining  (enlarged region). After 15  min, the structures stained by  GalNacT2–GFP and the vesicular structures stained with  the anti–Xklp3-tail antibody  are completely distinct (1  hour, enlarged region). (B)  XL177 cells were incubated  with 5 μg/ml of BFA for 5  min or 1 h and double stained  with the anti–Xklp3-tail and  the anti–KDEL-receptor antibodies. The KDEL-receptor and Xklp3 colocalize on  vesicular structures at early  time points (enlarged region,  overlay, 5 min). After 1 h of  treatment, vesicular structures positive for both Xklp3  and the KDEL-receptor are  still seen, but part of this  colocalization is lost (enlarged  region, overlay, 1 hour). Red,  Xklp3; green, corresponding  marker. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132969&req=5

Figure 5: Xklp3 and the KDEL receptor have a similar behavior upon BFA treatment. (A) A6 cells expressing Gal NacT2–GFP constitutively were incubated with 5 αg/ml of BFA for 5 min or 1 h and then stained with the anti– Xklp3-tail antibodies. After 5 min of BFA treatment, GalNacT2–GFP starts to be redistributed into the ER often appearing on tubular structures. The anti–Xklp3-tail antibody gives a punctuate staining dispersed in the cytoplasm, often associated to but not overlapping with the GalNacT2–GFP staining (enlarged region). After 15 min, the structures stained by GalNacT2–GFP and the vesicular structures stained with the anti–Xklp3-tail antibody are completely distinct (1 hour, enlarged region). (B) XL177 cells were incubated with 5 μg/ml of BFA for 5 min or 1 h and double stained with the anti–Xklp3-tail and the anti–KDEL-receptor antibodies. The KDEL-receptor and Xklp3 colocalize on vesicular structures at early time points (enlarged region, overlay, 5 min). After 1 h of treatment, vesicular structures positive for both Xklp3 and the KDEL-receptor are still seen, but part of this colocalization is lost (enlarged region, overlay, 1 hour). Red, Xklp3; green, corresponding marker. Bar, 10 μm.

Mentions: In a first attempt to determine whether Xklp3 was associated with Golgi stacks, we examined its relocalization after BFA treatment. BFA is used to discriminate secretory pathway subcompartments. This drug prevents the binding of peripheral coatomer proteins type 1 (COPI) proteins to Golgi membranes leading to membrane tubulation and redistribution of Golgi resident proteins into the ER (Doms et al., 1989; Scheel et al., 1997). However, some recycling proteins such as the KDEL-receptor, do not redistribute into the ER but are dispersed into vesicular structures throughout the cytoplasm (Tang et al., 1995; Fullekrüg et al., 1997). We verified that BFA treatment of Xenopus A6 and XL177 cells did result in a cytoplasmic redistribution of β-COP, a component of the COPI complex (data not shown). After 5 min of BFA treatment, Xklp3 was still present in some perinuclear structures and appeared in punctuate structures dispersed throughout the cytoplasm whereas the GalNacT2–GFP was associated with long tubular processes (Fig. 5 A). After 1 h of treatment, Xklp3 was associated with dispersed peripheral vesicular structures while GalNacT2–GFP had completely redistributed into the ER (Fig. 5 A). The lack of redistribution to the ER at later time points suggested that Xklp3 was not truly associated with the Golgi stacks. We then compared Xklp3 distribution to that of the KDEL receptor in BFA-treated cells (Fig. 5 B). During the first 30 min of BFA treatment, Xklp3 and the KDEL receptor colocalized on some vesicular structures (Fig. 5 B, 5 min, enlarged region). After 1 h of treatment, some of the vesicles stained by the anti-Xklp3 antibody were still positive for the KDEL receptor (Fig. 5 B, 1 hour, enlarged region) although a fraction of them were not. These results suggested that Xklp3 was associated with membrane structures distinct from the Golgi stacks and somehow related to the recycling compartment.


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 and the KDEL  receptor have a similar behavior upon BFA treatment.  (A) A6 cells expressing Gal  NacT2–GFP constitutively  were incubated with 5 αg/ml  of BFA for 5 min or 1 h and  then stained with the anti– Xklp3-tail antibodies. After  5 min of BFA treatment,  GalNacT2–GFP starts to be  redistributed into the ER often appearing on tubular  structures. The anti–Xklp3-tail antibody gives a punctuate staining dispersed in the  cytoplasm, often associated  to but not overlapping with  the GalNacT2–GFP staining  (enlarged region). After 15  min, the structures stained by  GalNacT2–GFP and the vesicular structures stained with  the anti–Xklp3-tail antibody  are completely distinct (1  hour, enlarged region). (B)  XL177 cells were incubated  with 5 μg/ml of BFA for 5  min or 1 h and double stained  with the anti–Xklp3-tail and  the anti–KDEL-receptor antibodies. The KDEL-receptor and Xklp3 colocalize on  vesicular structures at early  time points (enlarged region,  overlay, 5 min). After 1 h of  treatment, vesicular structures positive for both Xklp3  and the KDEL-receptor are  still seen, but part of this  colocalization is lost (enlarged  region, overlay, 1 hour). Red,  Xklp3; green, corresponding  marker. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Xklp3 and the KDEL receptor have a similar behavior upon BFA treatment. (A) A6 cells expressing Gal NacT2–GFP constitutively were incubated with 5 αg/ml of BFA for 5 min or 1 h and then stained with the anti– Xklp3-tail antibodies. After 5 min of BFA treatment, GalNacT2–GFP starts to be redistributed into the ER often appearing on tubular structures. The anti–Xklp3-tail antibody gives a punctuate staining dispersed in the cytoplasm, often associated to but not overlapping with the GalNacT2–GFP staining (enlarged region). After 15 min, the structures stained by GalNacT2–GFP and the vesicular structures stained with the anti–Xklp3-tail antibody are completely distinct (1 hour, enlarged region). (B) XL177 cells were incubated with 5 μg/ml of BFA for 5 min or 1 h and double stained with the anti–Xklp3-tail and the anti–KDEL-receptor antibodies. The KDEL-receptor and Xklp3 colocalize on vesicular structures at early time points (enlarged region, overlay, 5 min). After 1 h of treatment, vesicular structures positive for both Xklp3 and the KDEL-receptor are still seen, but part of this colocalization is lost (enlarged region, overlay, 1 hour). Red, Xklp3; green, corresponding marker. Bar, 10 μm.
Mentions: In a first attempt to determine whether Xklp3 was associated with Golgi stacks, we examined its relocalization after BFA treatment. BFA is used to discriminate secretory pathway subcompartments. This drug prevents the binding of peripheral coatomer proteins type 1 (COPI) proteins to Golgi membranes leading to membrane tubulation and redistribution of Golgi resident proteins into the ER (Doms et al., 1989; Scheel et al., 1997). However, some recycling proteins such as the KDEL-receptor, do not redistribute into the ER but are dispersed into vesicular structures throughout the cytoplasm (Tang et al., 1995; Fullekrüg et al., 1997). We verified that BFA treatment of Xenopus A6 and XL177 cells did result in a cytoplasmic redistribution of β-COP, a component of the COPI complex (data not shown). After 5 min of BFA treatment, Xklp3 was still present in some perinuclear structures and appeared in punctuate structures dispersed throughout the cytoplasm whereas the GalNacT2–GFP was associated with long tubular processes (Fig. 5 A). After 1 h of treatment, Xklp3 was associated with dispersed peripheral vesicular structures while GalNacT2–GFP had completely redistributed into the ER (Fig. 5 A). The lack of redistribution to the ER at later time points suggested that Xklp3 was not truly associated with the Golgi stacks. We then compared Xklp3 distribution to that of the KDEL receptor in BFA-treated cells (Fig. 5 B). During the first 30 min of BFA treatment, Xklp3 and the KDEL receptor colocalized on some vesicular structures (Fig. 5 B, 5 min, enlarged region). After 1 h of treatment, some of the vesicles stained by the anti-Xklp3 antibody were still positive for the KDEL receptor (Fig. 5 B, 1 hour, enlarged region) although a fraction of them were not. These results suggested that Xklp3 was associated with membrane structures distinct from the Golgi stacks and somehow related to the recycling compartment.

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