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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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Primary sequence, structural features of Xklp3 and sequence similarity to Kinesin II subfamily members. (A) Xklp3 protein sequence. Italics and  boldface, conserved regions corresponding to the sequence used for the design  of the degenerated oligos used in the original screen for Xenopus KLPs; double  underline, ATP-binding site residues; underline, kinesin motor conserved MT-binding site; dotted underline, region rich in charged amino acids that is present  in other members of the family. This region has been proposed to favor heterodimerization over homodimerization. These sequence  data are available from GenBank/EMBL/DDBJ under accession number AJ009839. (B) Output of the pepcoil program showing the  probability of formation of coiled-coil interactions. Below a schematic representation of Xklp3 is shown, with regions predicted to be  coiled coil (white). Asterisk, region rich in charged amino acids that interrupts the coiled-coil predicted domain and is present in other  members of the family. The ATP (dark line) and MT binding sites (white rectangle) in the motor region are indicated. (C) Similarity between protein sequences of Xklp3 and the other Kinesin II family members. Schematic representation of the Kinesin II heterotrimeric  complex, composed of two closely related kinesins (Klp1 and Klp2) and a third nonmotor subunit (KAP). Each kinesin-like protein has  three subdomains: motor (red), stalk (green), and tail (blue). The third nonmotor subunit (i.e., KAP115 in sea urchin, KAP3 in mouse) is  represented as a gray oval interacting with the tails. Xklp3 sequence is taken as a reference and the numbers correspond to the percentage of similarities found in the different domains between Xklp3 and the corresponding KLP. The first group comprises KLPs more  closely related to Xklp3, the second group comprises the second KLP subunit from the complex. In the third subgroup, Xklp3 sequence  is compared with the mouse KIF3C, a motor able to heterodimerize with KIF3A but not with KIF3B. We place other members from  this subfamily in the fourth group.
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Figure 1: Primary sequence, structural features of Xklp3 and sequence similarity to Kinesin II subfamily members. (A) Xklp3 protein sequence. Italics and boldface, conserved regions corresponding to the sequence used for the design of the degenerated oligos used in the original screen for Xenopus KLPs; double underline, ATP-binding site residues; underline, kinesin motor conserved MT-binding site; dotted underline, region rich in charged amino acids that is present in other members of the family. This region has been proposed to favor heterodimerization over homodimerization. These sequence data are available from GenBank/EMBL/DDBJ under accession number AJ009839. (B) Output of the pepcoil program showing the probability of formation of coiled-coil interactions. Below a schematic representation of Xklp3 is shown, with regions predicted to be coiled coil (white). Asterisk, region rich in charged amino acids that interrupts the coiled-coil predicted domain and is present in other members of the family. The ATP (dark line) and MT binding sites (white rectangle) in the motor region are indicated. (C) Similarity between protein sequences of Xklp3 and the other Kinesin II family members. Schematic representation of the Kinesin II heterotrimeric complex, composed of two closely related kinesins (Klp1 and Klp2) and a third nonmotor subunit (KAP). Each kinesin-like protein has three subdomains: motor (red), stalk (green), and tail (blue). The third nonmotor subunit (i.e., KAP115 in sea urchin, KAP3 in mouse) is represented as a gray oval interacting with the tails. Xklp3 sequence is taken as a reference and the numbers correspond to the percentage of similarities found in the different domains between Xklp3 and the corresponding KLP. The first group comprises KLPs more closely related to Xklp3, the second group comprises the second KLP subunit from the complex. In the third subgroup, Xklp3 sequence is compared with the mouse KIF3C, a motor able to heterodimerize with KIF3A but not with KIF3B. We place other members from this subfamily in the fourth group.

Mentions: The deduced protein sequence showed that Xklp3 had a conventional kinesin-like protein (KLP) organization with the NH2-terminal motor domain, an α-helical region predicted to be involved in coiled-coil interactions and a globular COOH-terminal tail domain (Fig. 1 A). Alignment of the Xklp3 motor domain sequence with other KLP sequences had previously shown that Xklp3 was closely related to Drosophila KLP4 and KLP5 (renamed KLP64D and KLP68D), both members of the KIF3/KRP85/95 or Kinesin II subfamily (Vernos et al., 1993; Pesavento et al., 1994; Scholey, 1996). Xklp3 full-length sequence showed that it also shared a high degree of similarity to these KLPs outside the motor domain. The highest scores were obtained with KIF3B (Yamazaki et al., 1995), with 90% amino acid similarity in the motor domain, 85% in the stalk, and 74% in the tail (Fig. 1 B). This high level of conservation suggests that Xklp3 is probably the Xenopus counterpart of mouse KIF3B.


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)

Primary sequence, structural features of Xklp3 and sequence similarity to Kinesin II subfamily members. (A) Xklp3 protein sequence. Italics and  boldface, conserved regions corresponding to the sequence used for the design  of the degenerated oligos used in the original screen for Xenopus KLPs; double  underline, ATP-binding site residues; underline, kinesin motor conserved MT-binding site; dotted underline, region rich in charged amino acids that is present  in other members of the family. This region has been proposed to favor heterodimerization over homodimerization. These sequence  data are available from GenBank/EMBL/DDBJ under accession number AJ009839. (B) Output of the pepcoil program showing the  probability of formation of coiled-coil interactions. Below a schematic representation of Xklp3 is shown, with regions predicted to be  coiled coil (white). Asterisk, region rich in charged amino acids that interrupts the coiled-coil predicted domain and is present in other  members of the family. The ATP (dark line) and MT binding sites (white rectangle) in the motor region are indicated. (C) Similarity between protein sequences of Xklp3 and the other Kinesin II family members. Schematic representation of the Kinesin II heterotrimeric  complex, composed of two closely related kinesins (Klp1 and Klp2) and a third nonmotor subunit (KAP). Each kinesin-like protein has  three subdomains: motor (red), stalk (green), and tail (blue). The third nonmotor subunit (i.e., KAP115 in sea urchin, KAP3 in mouse) is  represented as a gray oval interacting with the tails. Xklp3 sequence is taken as a reference and the numbers correspond to the percentage of similarities found in the different domains between Xklp3 and the corresponding KLP. The first group comprises KLPs more  closely related to Xklp3, the second group comprises the second KLP subunit from the complex. In the third subgroup, Xklp3 sequence  is compared with the mouse KIF3C, a motor able to heterodimerize with KIF3A but not with KIF3B. We place other members from  this subfamily in the fourth group.
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Related In: Results  -  Collection

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Figure 1: Primary sequence, structural features of Xklp3 and sequence similarity to Kinesin II subfamily members. (A) Xklp3 protein sequence. Italics and boldface, conserved regions corresponding to the sequence used for the design of the degenerated oligos used in the original screen for Xenopus KLPs; double underline, ATP-binding site residues; underline, kinesin motor conserved MT-binding site; dotted underline, region rich in charged amino acids that is present in other members of the family. This region has been proposed to favor heterodimerization over homodimerization. These sequence data are available from GenBank/EMBL/DDBJ under accession number AJ009839. (B) Output of the pepcoil program showing the probability of formation of coiled-coil interactions. Below a schematic representation of Xklp3 is shown, with regions predicted to be coiled coil (white). Asterisk, region rich in charged amino acids that interrupts the coiled-coil predicted domain and is present in other members of the family. The ATP (dark line) and MT binding sites (white rectangle) in the motor region are indicated. (C) Similarity between protein sequences of Xklp3 and the other Kinesin II family members. Schematic representation of the Kinesin II heterotrimeric complex, composed of two closely related kinesins (Klp1 and Klp2) and a third nonmotor subunit (KAP). Each kinesin-like protein has three subdomains: motor (red), stalk (green), and tail (blue). The third nonmotor subunit (i.e., KAP115 in sea urchin, KAP3 in mouse) is represented as a gray oval interacting with the tails. Xklp3 sequence is taken as a reference and the numbers correspond to the percentage of similarities found in the different domains between Xklp3 and the corresponding KLP. The first group comprises KLPs more closely related to Xklp3, the second group comprises the second KLP subunit from the complex. In the third subgroup, Xklp3 sequence is compared with the mouse KIF3C, a motor able to heterodimerize with KIF3A but not with KIF3B. We place other members from this subfamily in the fourth group.
Mentions: The deduced protein sequence showed that Xklp3 had a conventional kinesin-like protein (KLP) organization with the NH2-terminal motor domain, an α-helical region predicted to be involved in coiled-coil interactions and a globular COOH-terminal tail domain (Fig. 1 A). Alignment of the Xklp3 motor domain sequence with other KLP sequences had previously shown that Xklp3 was closely related to Drosophila KLP4 and KLP5 (renamed KLP64D and KLP68D), both members of the KIF3/KRP85/95 or Kinesin II subfamily (Vernos et al., 1993; Pesavento et al., 1994; Scholey, 1996). Xklp3 full-length sequence showed that it also shared a high degree of similarity to these KLPs outside the motor domain. The highest scores were obtained with KIF3B (Yamazaki et al., 1995), with 90% amino acid similarity in the motor domain, 85% in the stalk, and 74% in the tail (Fig. 1 B). This high level of conservation suggests that Xklp3 is probably the Xenopus counterpart of mouse KIF3B.

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