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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 localizes to  tubular-vesicular structures  between the ER and the  Golgi. (A) Double immunogold labeling of Xklp3  (mAb X3T-3A6, 10-nm gold)  and the KDEL-receptor (polyclonal anti-erd2, 5-nm gold)  on cryosections from A6-GalT–GFP cells. Xklp3 is  present on tubular-vesicular  structures (arrows and arrowheads) around the Golgi-cisternae (G) where it colocalizes with the KDEL-receptor  (arrows). Similar results are  obtained with the polyclonal  anti-tail (see Table I for  quantification). (B) Double  immunogold labeling of Xklp3  (monoclonal anti–Xklp3-tail,  mAb X3T-3A6, 10-nm gold)  and the GalT–GFP (polyclonal anti-GFP, 5-nm gold).  GalT–GFP is localized exclusively to the Golgi cisternae  and there is no clear colocalization with Xklp3 (arrowheads) When Xklp3 is found  on the Golgi stacks, it is always at its periphery, on budding-like structures (A–C).  Similar results are obtained  with the polyclonal anti-tail  (see Table I for quantification). (C) Double immunogold labeling of Xklp3  (polyclonal anti–Xklp3-tail,  10-nm gold, arrows and arrowheads) and PDI (monoclonal anti-PDI, 5-nm gold). The PDI is found in cisternae throughout the cytoplasm. Xklp3 is often seen on one side of these ER membrane structures (arrows) (see Table I for quantification). Bar, 100 nm.
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Figure 6: Xklp3 localizes to tubular-vesicular structures between the ER and the Golgi. (A) Double immunogold labeling of Xklp3 (mAb X3T-3A6, 10-nm gold) and the KDEL-receptor (polyclonal anti-erd2, 5-nm gold) on cryosections from A6-GalT–GFP cells. Xklp3 is present on tubular-vesicular structures (arrows and arrowheads) around the Golgi-cisternae (G) where it colocalizes with the KDEL-receptor (arrows). Similar results are obtained with the polyclonal anti-tail (see Table I for quantification). (B) Double immunogold labeling of Xklp3 (monoclonal anti–Xklp3-tail, mAb X3T-3A6, 10-nm gold) and the GalT–GFP (polyclonal anti-GFP, 5-nm gold). GalT–GFP is localized exclusively to the Golgi cisternae and there is no clear colocalization with Xklp3 (arrowheads) When Xklp3 is found on the Golgi stacks, it is always at its periphery, on budding-like structures (A–C). Similar results are obtained with the polyclonal anti-tail (see Table I for quantification). (C) Double immunogold labeling of Xklp3 (polyclonal anti–Xklp3-tail, 10-nm gold, arrows and arrowheads) and PDI (monoclonal anti-PDI, 5-nm gold). The PDI is found in cisternae throughout the cytoplasm. Xklp3 is often seen on one side of these ER membrane structures (arrows) (see Table I for quantification). Bar, 100 nm.

Mentions: To identify the nature of the Golgi elements to which Xklp3 was associated, we examined thin frozen sections of cells using immunogold EM. Previous EM studies have shown that the Golgi apparatus is composed of stacks of flattened cisternae enriched in glycosylating enzymes, and tubulovesicular structures associated with the rims of the stacks probably involved in specific transport steps (Rambourg and Clermont, 1996). We first looked at the relative localization of Xklp3 and the GalT–GFP chimera using the monoclonal anti–Xklp3-tail (mAb X3T-3A6) and a polyclonal anti-GFP antibody followed by detection with protein A coupled to 10-nm (Xklp3) and 5-nm (GFP) gold, respectively. GalT–GFP was only found in the stacked cisternae of the Golgi complex as expected (Fig. 6 B, 5-nm gold), whereas Xklp3 was associated with tubular-vesicular structures sometimes in the proximity of the stacks (Fig. 6, A and B, arrows and arrowheads). A similar experiment using a polyclonal anti–KDEL-receptor and the same anti Xklp3 antibodies revealed that Xklp3 (10-nm gold) was found on tubulovesicular structures positive for the KDEL-receptor (Fig 6 A, arrows) (Table I, quantification). The rest of Xklp3 was localized partly in the cytoplasm or associated with unlabeled membranous elements some of which were morphologically similar to the ER. A double-labeling experiment with an anti-PDI antibody showed Xklp3 was localized as well on structures bearing PDI (Fig. 6 C, arrows) (Table I, quantification). EM studies have shown that the KDEL-receptor is distributed among the cis-Golgi, tubular-vesicular elements of the intermediate compartment and ER membranes (Griffiths et al., 1994). Thus the distribution of Xklp3 is similar to that of an intermediate compartment protein.


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 localizes to  tubular-vesicular structures  between the ER and the  Golgi. (A) Double immunogold labeling of Xklp3  (mAb X3T-3A6, 10-nm gold)  and the KDEL-receptor (polyclonal anti-erd2, 5-nm gold)  on cryosections from A6-GalT–GFP cells. Xklp3 is  present on tubular-vesicular  structures (arrows and arrowheads) around the Golgi-cisternae (G) where it colocalizes with the KDEL-receptor  (arrows). Similar results are  obtained with the polyclonal  anti-tail (see Table I for  quantification). (B) Double  immunogold labeling of Xklp3  (monoclonal anti–Xklp3-tail,  mAb X3T-3A6, 10-nm gold)  and the GalT–GFP (polyclonal anti-GFP, 5-nm gold).  GalT–GFP is localized exclusively to the Golgi cisternae  and there is no clear colocalization with Xklp3 (arrowheads) When Xklp3 is found  on the Golgi stacks, it is always at its periphery, on budding-like structures (A–C).  Similar results are obtained  with the polyclonal anti-tail  (see Table I for quantification). (C) Double immunogold labeling of Xklp3  (polyclonal anti–Xklp3-tail,  10-nm gold, arrows and arrowheads) and PDI (monoclonal anti-PDI, 5-nm gold). The PDI is found in cisternae throughout the cytoplasm. Xklp3 is often seen on one side of these ER membrane structures (arrows) (see Table I for quantification). Bar, 100 nm.
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Related In: Results  -  Collection

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Figure 6: Xklp3 localizes to tubular-vesicular structures between the ER and the Golgi. (A) Double immunogold labeling of Xklp3 (mAb X3T-3A6, 10-nm gold) and the KDEL-receptor (polyclonal anti-erd2, 5-nm gold) on cryosections from A6-GalT–GFP cells. Xklp3 is present on tubular-vesicular structures (arrows and arrowheads) around the Golgi-cisternae (G) where it colocalizes with the KDEL-receptor (arrows). Similar results are obtained with the polyclonal anti-tail (see Table I for quantification). (B) Double immunogold labeling of Xklp3 (monoclonal anti–Xklp3-tail, mAb X3T-3A6, 10-nm gold) and the GalT–GFP (polyclonal anti-GFP, 5-nm gold). GalT–GFP is localized exclusively to the Golgi cisternae and there is no clear colocalization with Xklp3 (arrowheads) When Xklp3 is found on the Golgi stacks, it is always at its periphery, on budding-like structures (A–C). Similar results are obtained with the polyclonal anti-tail (see Table I for quantification). (C) Double immunogold labeling of Xklp3 (polyclonal anti–Xklp3-tail, 10-nm gold, arrows and arrowheads) and PDI (monoclonal anti-PDI, 5-nm gold). The PDI is found in cisternae throughout the cytoplasm. Xklp3 is often seen on one side of these ER membrane structures (arrows) (see Table I for quantification). Bar, 100 nm.
Mentions: To identify the nature of the Golgi elements to which Xklp3 was associated, we examined thin frozen sections of cells using immunogold EM. Previous EM studies have shown that the Golgi apparatus is composed of stacks of flattened cisternae enriched in glycosylating enzymes, and tubulovesicular structures associated with the rims of the stacks probably involved in specific transport steps (Rambourg and Clermont, 1996). We first looked at the relative localization of Xklp3 and the GalT–GFP chimera using the monoclonal anti–Xklp3-tail (mAb X3T-3A6) and a polyclonal anti-GFP antibody followed by detection with protein A coupled to 10-nm (Xklp3) and 5-nm (GFP) gold, respectively. GalT–GFP was only found in the stacked cisternae of the Golgi complex as expected (Fig. 6 B, 5-nm gold), whereas Xklp3 was associated with tubular-vesicular structures sometimes in the proximity of the stacks (Fig. 6, A and B, arrows and arrowheads). A similar experiment using a polyclonal anti–KDEL-receptor and the same anti Xklp3 antibodies revealed that Xklp3 (10-nm gold) was found on tubulovesicular structures positive for the KDEL-receptor (Fig 6 A, arrows) (Table I, quantification). The rest of Xklp3 was localized partly in the cytoplasm or associated with unlabeled membranous elements some of which were morphologically similar to the ER. A double-labeling experiment with an anti-PDI antibody showed Xklp3 was localized as well on structures bearing PDI (Fig. 6 C, arrows) (Table I, quantification). EM studies have shown that the KDEL-receptor is distributed among the cis-Golgi, tubular-vesicular elements of the intermediate compartment and ER membranes (Griffiths et al., 1994). Thus the distribution of Xklp3 is similar to that of an intermediate compartment protein.

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