A fission yeast kinesin affects Golgi membrane recycling

Abstract

We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3+, which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope‐tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule‐associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin‐based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4‐64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild‐type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi‐to‐ER recycling in wild‐type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild‐type strain. We conclude that Klp3 plays a role in BFA‐induced membrane transport. The nucleotide sequence of S. pombe klp3+ was submitted to GenBank under Accession No. AF154055. Copyright © 2000 John Wiley & Sons, Ltd.

hob1+, the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress

“…Endocytosis was measured by internalization of the fluorescent styryl dye FM4-64 (Molecular Probes) as described (Brazer et al, 2000;Routhier et al, 2001). Images were taken with a Nikon Eclipse TE300 microscope fitted with a Nikon Plan Fluor  100 objective, using a Toshiba 3CCD camera.…”

“…(e) Endocytosis assay. A fluorescent dye uptake assay was performed using the styryl dye FM4-64 as described (Brazer et al, 2000). Cells were incubated at 301C in the presence of FM4-64 and photographed at the times indicated.…”

pkl1+andklp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

“…S. pombe expresses one dynein heavy chain (Yamamoto et al, 1999), and although this motor is essential for nuclear migration during the sexual phase of the life cycle, it plays no detectable role in mitosis. Klp3p (Brazer et al, 2000) and Klp4p, also known as Tea2p (Browning et al, 2000) are cytoplasmic motors in S. pombe that have no detectable role in mitosis, nor are their cytoplasmic functions essential. Given the plethora of motors in other organisms and the fact that several organisms have more than one KLP from the KAR3 subfamily (reviewed in Endow, 1999), we have sought additional KLPs in fission yeast that might function together with Cut7p and Pkl1p in mitosis.…”

Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic spindle poles

“…Mitochondria colocalize with microtubules during interphase of the fission yeast cell cycle (4,5), and certain mutations in tubulin genes cause dramatic changes in mitochondrial distribution in mutant S. pombe cells, with mitochondria becoming aggregated and asymmetrically positioned (4). However, mitochondrial distribution in fission yeast does not appear to depend on the activity of klp3, the major cytoplasmic kinesin (6), or the function of other kinesin-like proteins (B. A. Weir and M.P.Y., unpublished observations).…”