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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 is associated to heavy membranes. (A) Xklp3 is  found in a membrane fraction prepared from egg extracts. Western blot of a high-speed cytosol and the corresponding membrane fraction with the anti–Xklp3-tail antibody. Equivalent volumes from each fraction were loaded on each lane. (B) The  membrane fraction shown in A was resuspended in 2 M sucrose,  loaded at the bottom of a sucrose step gradient, and then centrifuged to equilibrium. Equivalent volumes of the different fractions were loaded on PAGE and transferred to nitrocellulose.  The Western blot was probed with the anti–Xklp3-tail antibody.  (1) 2 M/1.3 M sucrose (Bottom); (2) 1.3 M/0.86 M (heavy membranes, ∼ER); (3) 0.86 M/0.5 M (membranes ∼heavy Golgi); (4)  0.5 M/0.25 M (light membranes). (C) A fraction of Xklp3 is associated to heavy membranes from A6 cells extract. Cytosol (PNS)  was prepared from A6 cells expressing GFP–mannosidase II constitutively as described in Materials and Methods. The PNS was  then loaded on a sucrose step gradient (sucrose concentrations  are written above fraction numbers; arrows, interface between  two sucrose concentrations). Fractions were collected and run on  PAGE. The Western blot was probed sequentially with the following antibodies: anti–Xklp3-tail, anti-PDI (as an ER marker),  anti-GFP (to detect the Golgi marker mannosidase II–GFP), and  anti-tubulin (as a nonmembrane-associated molecule). Xklp3 is  present in soluble fractions 1–4. In addition, there are two peaks  of Xklp3 in fractions where heavy membranes sediment: one  around fraction 7 corresponding to membranes enriched in mannosidase II–GFP, and another around fraction 9–10 corresponding to membranes enriched in PDI.
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Figure 7: Xklp3 is associated to heavy membranes. (A) Xklp3 is found in a membrane fraction prepared from egg extracts. Western blot of a high-speed cytosol and the corresponding membrane fraction with the anti–Xklp3-tail antibody. Equivalent volumes from each fraction were loaded on each lane. (B) The membrane fraction shown in A was resuspended in 2 M sucrose, loaded at the bottom of a sucrose step gradient, and then centrifuged to equilibrium. Equivalent volumes of the different fractions were loaded on PAGE and transferred to nitrocellulose. The Western blot was probed with the anti–Xklp3-tail antibody. (1) 2 M/1.3 M sucrose (Bottom); (2) 1.3 M/0.86 M (heavy membranes, ∼ER); (3) 0.86 M/0.5 M (membranes ∼heavy Golgi); (4) 0.5 M/0.25 M (light membranes). (C) A fraction of Xklp3 is associated to heavy membranes from A6 cells extract. Cytosol (PNS) was prepared from A6 cells expressing GFP–mannosidase II constitutively as described in Materials and Methods. The PNS was then loaded on a sucrose step gradient (sucrose concentrations are written above fraction numbers; arrows, interface between two sucrose concentrations). Fractions were collected and run on PAGE. The Western blot was probed sequentially with the following antibodies: anti–Xklp3-tail, anti-PDI (as an ER marker), anti-GFP (to detect the Golgi marker mannosidase II–GFP), and anti-tubulin (as a nonmembrane-associated molecule). Xklp3 is present in soluble fractions 1–4. In addition, there are two peaks of Xklp3 in fractions where heavy membranes sediment: one around fraction 7 corresponding to membranes enriched in mannosidase II–GFP, and another around fraction 9–10 corresponding to membranes enriched in PDI.

Mentions: Membranes from the Golgi stacks, the ER, and recycling compartments behave differently upon sedimentation or floatation on sucrose gradients. We first separated the membranes from cytosolic proteins in egg extracts using a one step centrifugation, and further fractionated them by floatation. Approximately 20–25% of Xklp3 cosedimented with the membrane fraction (Fig. 7 A). We found Xklp3 associated with membranes that floated at the 1.3 M–0.86 M sucrose interface (like ER membranes) and at the 0.86 M–0.5 M sucrose interface (like Golgi membranes, Fig. 7 B). To further characterize the membrane fractions with which Xklp3 cosedimented, we prepared PNS from A6 cells stably transfected with MannII–GFP. The homogenates were then loaded on a sucrose step gradient (Fig. 7 C). Xklp3 was found in large amounts at the 0.25 M–0.5 M sucrose interface, probably corresponding to the soluble form of the trimeric complex. In addition, Xklp3 was found in two heavier fractions, at the 0.5 M–0.86 M sucrose interface which corresponded to a peak of Golgi membranes as revealed by the presence of MannII–GFP (Fig. 7 C, fraction 7, Xklp3, MannII–GFP) and at the 0.86 M–1.3 M sucrose interface which corresponded to a peak of ER/intermediate compartment as revealed by the presence of PDI. These results indicated that part of Xklp3 was associated with heavy membranes from both Golgi and ER/intermediate 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 is associated to heavy membranes. (A) Xklp3 is  found in a membrane fraction prepared from egg extracts. Western blot of a high-speed cytosol and the corresponding membrane fraction with the anti–Xklp3-tail antibody. Equivalent volumes from each fraction were loaded on each lane. (B) The  membrane fraction shown in A was resuspended in 2 M sucrose,  loaded at the bottom of a sucrose step gradient, and then centrifuged to equilibrium. Equivalent volumes of the different fractions were loaded on PAGE and transferred to nitrocellulose.  The Western blot was probed with the anti–Xklp3-tail antibody.  (1) 2 M/1.3 M sucrose (Bottom); (2) 1.3 M/0.86 M (heavy membranes, ∼ER); (3) 0.86 M/0.5 M (membranes ∼heavy Golgi); (4)  0.5 M/0.25 M (light membranes). (C) A fraction of Xklp3 is associated to heavy membranes from A6 cells extract. Cytosol (PNS)  was prepared from A6 cells expressing GFP–mannosidase II constitutively as described in Materials and Methods. The PNS was  then loaded on a sucrose step gradient (sucrose concentrations  are written above fraction numbers; arrows, interface between  two sucrose concentrations). Fractions were collected and run on  PAGE. The Western blot was probed sequentially with the following antibodies: anti–Xklp3-tail, anti-PDI (as an ER marker),  anti-GFP (to detect the Golgi marker mannosidase II–GFP), and  anti-tubulin (as a nonmembrane-associated molecule). Xklp3 is  present in soluble fractions 1–4. In addition, there are two peaks  of Xklp3 in fractions where heavy membranes sediment: one  around fraction 7 corresponding to membranes enriched in mannosidase II–GFP, and another around fraction 9–10 corresponding to membranes enriched in PDI.
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Related In: Results  -  Collection

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Figure 7: Xklp3 is associated to heavy membranes. (A) Xklp3 is found in a membrane fraction prepared from egg extracts. Western blot of a high-speed cytosol and the corresponding membrane fraction with the anti–Xklp3-tail antibody. Equivalent volumes from each fraction were loaded on each lane. (B) The membrane fraction shown in A was resuspended in 2 M sucrose, loaded at the bottom of a sucrose step gradient, and then centrifuged to equilibrium. Equivalent volumes of the different fractions were loaded on PAGE and transferred to nitrocellulose. The Western blot was probed with the anti–Xklp3-tail antibody. (1) 2 M/1.3 M sucrose (Bottom); (2) 1.3 M/0.86 M (heavy membranes, ∼ER); (3) 0.86 M/0.5 M (membranes ∼heavy Golgi); (4) 0.5 M/0.25 M (light membranes). (C) A fraction of Xklp3 is associated to heavy membranes from A6 cells extract. Cytosol (PNS) was prepared from A6 cells expressing GFP–mannosidase II constitutively as described in Materials and Methods. The PNS was then loaded on a sucrose step gradient (sucrose concentrations are written above fraction numbers; arrows, interface between two sucrose concentrations). Fractions were collected and run on PAGE. The Western blot was probed sequentially with the following antibodies: anti–Xklp3-tail, anti-PDI (as an ER marker), anti-GFP (to detect the Golgi marker mannosidase II–GFP), and anti-tubulin (as a nonmembrane-associated molecule). Xklp3 is present in soluble fractions 1–4. In addition, there are two peaks of Xklp3 in fractions where heavy membranes sediment: one around fraction 7 corresponding to membranes enriched in mannosidase II–GFP, and another around fraction 9–10 corresponding to membranes enriched in PDI.
Mentions: Membranes from the Golgi stacks, the ER, and recycling compartments behave differently upon sedimentation or floatation on sucrose gradients. We first separated the membranes from cytosolic proteins in egg extracts using a one step centrifugation, and further fractionated them by floatation. Approximately 20–25% of Xklp3 cosedimented with the membrane fraction (Fig. 7 A). We found Xklp3 associated with membranes that floated at the 1.3 M–0.86 M sucrose interface (like ER membranes) and at the 0.86 M–0.5 M sucrose interface (like Golgi membranes, Fig. 7 B). To further characterize the membrane fractions with which Xklp3 cosedimented, we prepared PNS from A6 cells stably transfected with MannII–GFP. The homogenates were then loaded on a sucrose step gradient (Fig. 7 C). Xklp3 was found in large amounts at the 0.25 M–0.5 M sucrose interface, probably corresponding to the soluble form of the trimeric complex. In addition, Xklp3 was found in two heavier fractions, at the 0.5 M–0.86 M sucrose interface which corresponded to a peak of Golgi membranes as revealed by the presence of MannII–GFP (Fig. 7 C, fraction 7, Xklp3, MannII–GFP) and at the 0.86 M–1.3 M sucrose interface which corresponded to a peak of ER/intermediate compartment as revealed by the presence of PDI. These results indicated that part of Xklp3 was associated with heavy membranes from both Golgi and ER/intermediate 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