Role of type I myosins in receptor-mediated endocytosis in yeast

Science

Volumn 272, Issue 5261, 1996, Pages 533-535

  Geli, M Isabel ^a   Riezman, Howard ^a  

^a UNIVERSITY OF BASEL   (Switzerland)

Author keywords

[No Author keywords available]

Indexed keywords

MYOSIN;

ARTICLE; ENDOCYTOSIS; FUNGAL GENETICS; FUNGUS GROWTH; FUNGUS MUTANT; GENE DELETION; NONHUMAN; PRIORITY JOURNAL; TEMPERATURE SENSITIVE MUTANT; YEAST;

EID: 0029923034     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5261.533     Document Type: Article

References (33)

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12. MYO3 and MYO5 were deleted in the same diploid to obtain a heterozygotic strain, RH3375 (MATa/ MATα ade2/ADE2 his3/his3 myo3Δ::HIS3/MYO3 myo5Δ::TRP1/MYO5 leu2/leu2 lys2/LYS2 trp1/ trp1 ura3/ura3 bar1/bar1). From this strain the RH3376, RH3377, and RH3378 strains were generated by tetrad dissection (Table 1). Deletion of MYO3 has been described (6). For deletion of MYO5, the fragments Eco RI-Hind III and Pst I-Bgl II (Fig 1A) were used to flank a 0.9-kb DNA fragment carrying the TRP1 gene. The spores were analyzed by replica plating on synthetic dextrose medium lacking the corresponding amino acids. None of the predicted double mutants (TRP1 HIS3) out of 20 dissected tetrads formed colonies. A double mutant harboring plasmid pmyo5-1 or pMYO5 (13) was able to form pinpoint-sized colonies upon plasmid loss. When the pmyo5-1-carrying strain was shifted to 37°C, the proportion of unbudded cells did not change
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16. E511 (Glu511) of Myo2p in the putative actin binding domain [G. C. Johnston, J. A. Pendergast, R. A Singer, J. Cell Biol. 113, 539 (1991)] corresponds to E472 (Glu472) in Myo5p (Fig. 1A). The allele E472K (Glu472 → Lys472) (myo5-1) was generated by PCR and cloned into a centromere-based plasmid [YC-plac33; Gene 74, 527 (1938)] to generate pmyo5-1. Wild-type MYO5 was also cloned into YCplac33 to generate pMYO5.
17. E511 (Glu511) of Myo2p in the putative actin binding domain [G. C. Johnston, J. A. Pendergast, R. A Singer, J. Cell Biol. 113, 539 (1991)] corresponds to E472 (Glu472) in Myo5p (Fig. 1A). The allele E472K (Glu472 → Lys472) (myo5-1) was generated by PCR and cloned into a centromere-based plasmid [YC-plac33; Gene 74, 527 (1938)] to generate pmyo5-1. Wild-type MYO5 was also cloned into YCplac33 to generate pMYO5.
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33. We thank Riezman laboratory members for discussion; S. Schröder, A L. Munn, A. Wesp, C. Sutterlin, and M. Schönbächler for critical reading of the manuscript; T. Aust and N. Stern for technical assistance, and H. Goodson for the plasmid to delete MYO3. Funded by a grant from the Swiss National Science Foundation (to H.R.). M.I.G. was supported by a European Molecular Biology Organization long-term fellowship.