Molecular motor KIF1C is not essential for mouse survival and motor-dependent retrograde Golgi apparatus-to-endoplasmic reticulum transport - PubMed (original) (raw)

Molecular motor KIF1C is not essential for mouse survival and motor-dependent retrograde Golgi apparatus-to-endoplasmic reticulum transport

Kazuo Nakajima et al. Mol Cell Biol. 2002 Feb.

Abstract

KIF1C is a new member of the kinesin superfamily of proteins (KIFs), which act as microtubule-based molecular motors involved in intracellular transport. We cloned full-length mouse kif1C cDNA, which turned out to have a high homology to a mitochondrial motor KIF1Balpha and to be expressed ubiquitously. To investigate the in vivo significance of KIF1C, we generated kif1C(-/-) mice by knocking in the beta-galactosidase gene into the motor domain of kif1C gene. On staining of LacZ, we detected its expression in the heart, liver, hippocampus, and cerebellum. Unexpectedly, kif1C(-/-) mice were viable and showed no obvious abnormalities. Because immunocytochemistry showed partial colocalization of KIF1C with the Golgi marker protein, we compared the organelle distribution in primary lung fibroblasts from kif1C(+/+) and kif1C(-/-) mice. We found that there was no significant difference in the distribution of the Golgi apparatus or in the transport from the Golgi apparatus to the endoplasmic reticulum (ER) facilitated by brefeldin A between the two cells. This retrograde membrane transport was further confirmed to be normal by time-lapse analysis. Consequently, KIF1C is dispensable for the motor-dependent retrograde transport from the Golgi apparatus to the ER.

PubMed Disclaimer

Figures

FIG. 1.

Molecular cloning of the mouse kif1C gene. (A) Amino acid sequence alignment of the mammalian KIF1 subfamily members. Amino acids are numbered on the left and right sides of the sequence. Asterisks indicate identical amino acids, and dots show similar residues among four proteins. The kinesin head domain, which is conserved between conventional kinesin and the KIF1 family, is boxed. Major consensus sequences of KIFs are shown in boldface type. The sequence of mKIF1A was truncated at aa 666, where an arrowhead shows the end of the conserved head domain among the KIF1 family. The AF6/cno domain is underlined. The FHA domain is shaded. The sequence against which the antibody was raised is doubly underlined. The alignment was performed using the ClustalW algorithm and manually modified. (B) Ubiquitous expression pattern of KIF1C protein in mouse tissues (40 μg of protein was loaded in each lane). (C) Subcellular fractionation of the MDCK cells monitored for KIF1C protein. KIF1C was most abundant in the high-speed membrane-pellet fraction (100,000 × g precipitate [ppt]). (D) Nucleotide-dependent binding activity of KIF1C to microtubules. The AMP-PNP pellet was resuspended in ATP-containing (0, 5, and 10 mM) buffer and centrifuged. The pellets (P) and the supernatants (S) were analyzed by immunoblotting. CE, crude extract. Note that KIF1C was released from microtubules depending on the concentration of ATP.

FIG. 2.

Targeted disruption of the mouse kif1C gene. (A) Schematic drawing of the targeting strategy for kif1C. A 2.6-kb region (corresponding to aa 36 to 240) encoding the ATP binding motif P-loop was deleted and replaced with an SA-IRESβgeo cassette. A poly(A)-less diphtheria toxin A fragment (DT-A) was used as a negative selection marker. The probe used for screening the homologous recombination is also indicated. ATG, kif1C gene translation start site; P, _Pst_I; B, _Bam_HI; S, _Sma_I; Sp, _Spe_I; SA, splice acceptor sequence; WT, wild type; KO, knockout, (B) Southern blot analysis of genomic DNA isolated from mouse tail tissue. Genomic DNA was digested with _Pst_I and probed with a 240-bp fragment 5′ to the exon, yielding 5.0-kb restriction fragments from kif1C+/+ alleles and 1.6-kb fragments from _kif1C_−/− alleles. (C) Immunoblot analysis of KIF1C expression in whole-brain homogenates (30 μg of protein) of respective genotypes with anti-KIF1C antibody. No bands were detected in the _kif1C_−/− samples.

FIG. 3.

Histological examination of the knockout mice. (A to D) Detection of lacZ gene expression from kif1C+/− mice. (A) Heart. Bar, 100 μm. (B) Coronal section of hippocampal region in brain. Py, pyramidal layer in hippocampus. Bar, 500 μm. (C) Liver. Bar, 100 μm. (D) Sagittal section of cerebellum. ML, molecular layer; PL, Purkinje cell layer; GL, granular layer. Bar, 100 μm. (E to J) Histological examination. (E, G, and I) kif1C+/+ tissues; (F, H, and J) _kif1C_−/− tissues. There were no obvious structural changes in the tissues of the _kif1C_−/− mice. (E and F) Heart (hematoxylin and eosin stain). Bar, 10 μm. (G and H) Lung (hematoxylin and eosin stain). Bar, 200 μm. (I and J) Hippocampus (Bodian silver stain). Bar, 500 μm. DG, dentate gyrus; CA1, CA1 field; CA3, CA3 field.

FIG. 4.

Subcellular localization of KIF1C. (A) KIF1C signal in mouse liver. Note that the knockout tissue eliminated the signal (left, bottom). At higher magnification, the signal was localized in the perinuclear region of wild-type cells (right). N, nucleus. Bars, 200 μm (left) and 10 μm (right). (B) Intracellular localization of KIF1C. Fibroblasts were doubly stained with anti-Golgi CTR433 [red (a)] and anti-KIF1C [green (b)] antibodies. These two signals were mainly colocalized (C). Bar, 10 μm.

FIG. 5.

Distribution and BFA-induced breakdown of the Golgi apparatus. (A) Comparison of the immunostaining pattern using antibodies against Golgi marker proteins. There were no significant differences in staining pattern between kif1C+/+ and _kif1C_−/− cells. Bars, 25 μm (top) and 50 μm (bottom). (B) Disassembly of the Golgi apparatus facilitated by BFA. Cultured fibroblasts from kif1C+/+ and _kif1C_−/− mice were immunostained for the Golgi marker protein p58 after the treatment of cells with BFA (5 μg/ml) for 10 min at 37°C. Golgi disassembly was observed in both types of cells. Bar, 50 μm.

FIG. 6.

Golgi membrane tubule dynamics during the BFA-induced Golgi disassembly process. Fibroblasts were transfected with a pEYFP-galactosyltransferase fusion protein gene construct. Time-lapse images were processed after treatment with BFA (10 μg/ml) at 30°C. Tubule formation from the Golgi apparatus was observed in each genotype. The intervals between the top (A and C) and bottom (B and D) frames were 20 s. Arrows indicate tips of growing tubules. Arrowheads indicate detaching tubules. Bar, 5 μm.

References

1. Advani, R. J., H. R. Bae, J. B. Bock, D. S. Chao, Y. C. Doung, R. Prekeris, J. S. Yoo, and R. H. Scheller. 1998. Seven novel mammalian SNARE proteins localize to distinct membrane compartments. J. Biol. Chem. 273:10317–10324. - PubMed
1. Aizawa, H., Y. Sekine, R. Takemura, Z. Zhang, M. Nangaku, and N. Hirokawa. 1992. Kinesin family in murine central nervous system. J. Cell Biol. 119:1287–1296. - PMC - PubMed
1. Bloom, G. S., and T. A. Brashear. 1989. A novel 58-kDa protein associates with the Golgi apparatus and microtubules. J. Biol. Chem. 264:16083–16092. - PubMed
1. Bodian, P. 1936. A new method for staining nerve fibers and nerve ending in mounted paraffin sections. Anat. Rec. 65:89–96.
1. Dorner, C., T. Ciossek, S. Muller, P. H. Moller, A. Ullrich, and R. Lammers. 1998. Characterization of KIF1C, a new kinesin-like protein involved in vesicle transport from the Golgi apparatus to the endoplasmic reticulum. J. Biol. Chem. 273:20267–20275. - PubMed

Molecular motor KIF1C is not essential for mouse survival and motor-dependent retrograde Golgi apparatus-to-endoplasmic reticulum transport - PubMed (original) (raw)