Degradation of serotonin N-acetyltransferase, a circadian regulator, by the N-end rule pathway

Journal of Biological Chemistry

Volumn 291, Issue 33, 2016, Pages 17178-17196

  Wadas, Brandon ^a   Borjigin, Jimo ^b   Huang, Zheping ^c   Oh, Jang Hyun ^a   Hwang, Cheol Sang ^d   Varshavsky, Alexander ^a  

^a California Institute of Technology   (United States)

^b UNIVERSITY OF MICHIGAN MEDICAL SCHOOL   (United States)

^c UNIVERSITY OF CONNECTICUT SCHOOL OF MEDICINE   (United States)

^d Pohang University of Science and Technology (POSTECH)   (South Korea)

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACIDS; YEAST;

MAMMALIAN CELLS; N-ACETYLTRANSFERASES; N-TERMINAL SEQUENCES; N-TERMINALS; POLYUBIQUITYLATION; YEAST SACCHAROMYCES CEREVISIAE;

RATS;

ARALKYLAMINE ACETYLTRANSFERASE; AANAT PROTEIN, HUMAN; AANAT PROTEIN, RAT;

AMINO TERMINAL SEQUENCE; ARTICLE; CONTROLLED STUDY; EMBRYO; ENZYME DEGRADATION; ENZYME REGULATION; HUMAN; HUMAN CELL; MOLECULAR RECOGNITION; NONHUMAN; PRIORITY JOURNAL; SEQUENCE ANALYSIS; UBIQUITINATION; WILD TYPE; ACETYLATION; ANIMAL; GENETICS; HEK293 CELL LINE; METABOLISM; MUTATION; PHYSIOLOGY; PROTEIN DEGRADATION; RAT; SACCHAROMYCES CEREVISIAE;

ACETYLATION; ANIMALS; ARYLALKYLAMINE N-ACETYLTRANSFERASE; HEK293 CELLS; HUMANS; MUTATION; PROTEOLYSIS; RATS; SACCHAROMYCES CEREVISIAE; UBIQUITINATION;

EID: 84981350332     PISSN: 00219258     EISSN: 1083351X     Source Type: Journal    
DOI: 10.1074/jbc.M116.734640     Document Type: Article

References (102)

1. Klein, D. C. (2006) Evolution of the vertebrate pineal gland: the AANAT hypothesis. Chronobiol. Int. 23, 5-20
2. Coon, S. L., and Klein, D. C. (2006) Evolution of arylalkylamine N-acetyltransferase: emergence and divergence. Mol. Cell. Endocrinol. 252, 2-10
3. Li, J., You, X., Bian, C., Yu, H., Coon, S. L., and Shi, Q. (2016) Molecular evolution of aralkylamine N-acetyltransferase in fish: a genomic survey. Int. J. Mol. Sci. 17, E51
4. Borjigin, J., Zhang, L. S., and Calinescu, A. A. (2012) Circadian regulation of pineal gland rhythmicity. Mol. Cell. Endocrinol. 349, 13-19
5. Choi, B. H., Chae, H. D., Park, T. J., Oh, J., Lim, J., Kang, S. S., Ha, H., and Kim, K. T. (2004) Protein kinase C regulates the activity and stability of serotonin N-acetyltransferase. J. Neurochem. 90, 442-454
6. Szewczuk, L. M., Tarrant, M. K., Sample, V., Drury, W. J., 3rd, Zhang, J., and Cole, P. A. (2008) Analysis of serotonin N-acetyltransferase regulation in vitro and in live cells using protein semisynthesis. Biochemistry 47, 10407-10419
7. Obsil, T., Ghirlando, R., Klein, D. C., Ganguly, S., and Dyda, F. (2001) rystal structure of the 14-3-3ζ:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation. Cell 105, 257-267
8. Hickman, A. B., Namboodiri, M. A., Klein, D. C., and Dyda, F. (1999) The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 Å resolution with a bisubstrate analog. Cell 97, 361-369
9. Hickman, A. B., Klein, D. C., and Dyda, F. (1999) Melatonin biosynthesis: the structure of serotonin N-acetyltransferase at 2.5 Å resolution suggests a catalytic mechanism. Mol. Cell 3, 23-32
10. Borjigin, J., Wang, M. M., and Snyder, S. H. (1995) Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland. Nature 378, 783-785
11. Coon, S. L., Roseboom, P. H., Baler, R., Weller, J. L., Namboodiri, M. A., Koonin, E. V., and Klein, D. C. (1995) Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis. Science 270, 1681-1683
12. Tosini, G., Ye, K., and Iuvone, P. M. (2012) N-Acetylserotonin: neuroprotection, neurogenesis, and the sleepy brain. Neuroscientist 18, 645- 653
13. Jang, S. W., Liu, X., Pradoldej, S., Tosini, G., Chang, Q., Iuvone, P. M., and Ye, K. (2010) N-Acetylserotonin activates TrkB receptor in a circadian rhythm. Proc. Natl. Acad. Sci. U.S.A. 107, 3876-3881
14. Sompol, P., Liu, X., Baba, K., Paul, K. N., Tosini, G., Iuvone, P. M., and Ye, K. (2011) N-Acetylserotonin promotes hippocampal neuroprogenitor ell proliferation in sleep-deprived mice. Proc. Natl. Acad. Sci. U.S.A. 108, 8844-8849
15. Schippers, K. J., and Nichols, S. A. (2014) Deep, dark secrets of melatonin in animal evolution. Cell 159, 9-10
16. Zhdanova, I. V., Wang, S. Y., Leclair, O. U., and Danilova, N. P. (2001) elatonin promotes sleep-like state in zebrafish. Brain Res. 903, 263-268
17. Gandhi, A. V., Mosser, E. A., Oikonomou, G., and Prober, D. A. (2015) Melatonin is required for the circadian regulation of sleep. Neuron 85, 1193-1199
18. González, S., Moreno-Delgado, D., Moreno, E., Pérez-Capote, K., Franco, R., Mallol, J., Cortés, A., Casadó, V., Lluís, C., Ortiz, J., Ferré, S., Canela, E., and McCormick, P. J. (2012) Circadian-related heteromerization of adrenergic and dopamine D(4) receptors modulates melatonin synthesis and release in the pineal gland. PLoS Biol. 10, e1001347
19. Rawashdeh, O., and Maronde, E. (2012) The hormonal Zeitgeber melatonin: role as a circadian modulator in memory processing. Front. Mol. Neurosci. 5, 27
20. Arendt, J. (2006) Melatonin and human rhythms. Chronobiol. Int. 23, 21-37
21. Liu, T., and Borjigin, J. (2005) N-Acetyltransferase is not the rate-limiting enzyme of melatonin synthesis at night. J. Pineal Res. 39, 91-96
22. Tu, B. P., and McKnight, S. L. (2006) Metabolic cycles as an underlying basis of biological oscillations. Nat. Rev. Mol. Cell Biol. 7, 696-701
23. Masri, S., and Sassone-Corsi, P. (2013) The circadian clock: a framework linking metabolism, epigenetics and neuronal function. Nat. Rev. Neurosci. 14, 69-75
24. Edgar, R. S., Green, E. W., Zhao, Y., van Ooijen, G., Olmedo, M., Qin, X., Xu, Y., Pan, M., Valekunja, U. K., Feeney, K. A., Maywood, E. S., Hastings, M. H., Baliga, N. S., Merrow, M., Millar, A. J., Johnson, C. H., Kyriacou, C. P., O'Neill, J. S., and Reddy, A. B. (2012) Peroxiredoxins are conserved markers of circadian rhythms. Nature 485, 459- 464
25. Borjigin, J., Li, X., and Snyder, S. H. (1999) The pineal gland and melatonin: molecular and pharmacologic regulation. Annu. Rev. Pharmacol. Toxicol. 39, 53-65
26. Stehle, J. H., Saade, A., Rawashdeh, O., Ackermann, K., Jilg, A., Sebestény, T., and Maronde, E. (2011) A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases. J. Pineal Res. 51, 17- 43
27. Mohawk, J. A., and Takahashi, J. S. (2011) Cell autonomy and synchrony of suprachiasmatic nucleus circadian oscillators. Trends Neurosci. 34, 349-358
28. Ganguly, S., Gastel, J. A., Weller, J. L., Schwartz, C., Jaffe, H., Namboodiri, M. A., Coon, S. L., Hickman, A. B., Rollag, M., Obsil, T., Beauverger, P., Ferry, G., Boutin, J. A., and Klein, D. C. (2001) Role of a pineal cAMPoperated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis. Proc. Natl. Acad. Sci. U.S.A. 98, 8083-8088
29. Mukherjee, S., and Maitra, S. K. (2015) Gut melatonin in vertebrates: chronobiology and physiology. Front. Endocrinol. 6, 112
30. Hardeland, R. (2010) Melatonin metabolism in the central nervous system. Curr. Neuropharmacol. 8, 168-181
31. Chattoraj, A., Liu, T., Zhang, L. S., Huang, Z., and Borjigin, J. (2009) elatonin formation in mammals: in vivo perspectives. Rev. Endocr. Metab. Disord. 10, 237-243
32. Schomerus, C., Laedtke, E., Olcese, J., Weller, J. L., Klein, D. C., and Korf, H. W. (2002) Signal transduction and regulation of melatonin synthesis in bovine pinealocytes: impact of adrenergic, peptidergic and cholinergic stimuli. Cell Tissue Res. 309, 417- 428
33. Schomerus, C., Korf, H. W., Laedtke, E., Weller, J. L., and Klein, D. C. (2000) Selective adrenergic/cyclic AMP-dependent switch-off of proteasomal proteolysis alone switches on neural signal transduction: an example from the pineal gland. J. Neurochem. 75, 2123-2132
34. Bernard, M., Iuvone, P. M., Cassone, V. M., Roseboom, P. H., Coon, S. L., and Klein, D. C. (1997) Avian melatonin synthesis: photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina. J. Neurochem. 68, 213-224
35. Gastel, J. A., Roseboom, P. H., Rinaldi, P. A., Weller, J. L., and Klein, D. C. (1998) Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation. Science 279, 1358-1360
36. Roseboom, P. H., Coon, S. L., Baler, R., McCune, S. K., Weller, J. L., and Klein, D. C. (1996) Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland. Endocrinology 137, 3033-3045
37. Huang, Z., Liu, T., and Borjigin, J. (2010) N-terminal residues regulate proteasomal degradation of AANAT. J. Pineal Res. 48, 290-296
38. Bachmair, A., Finley, D., and Varshavsky, A. (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179-186
39. Hwang, C. S., Shemorry, A., and Varshavsky, A. (2010) N-terminal acetylation of cellular proteins creates specific degradation signals. Science 327, 973-977
40. Kim, H. K., Kim, R. R., Oh, J. H., Cho, H., Varshavsky, A., and Hwang, C. S. (2014) The N-terminal methionine of cellular proteins as a degradation signal. Cell 156, 158-169
41. Varshavsky, A. (2011) The N-end rule pathway and regulation by proteolysis. Protein Sci. 20, 1298-1345
42. Gibbs, D. J., Bacardit, J., Bachmair, A., and Holdsworth, M. J. (2014) The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions. Trends Cell Biol. 24, 603-611
43. Tasaki, T., Sriram, S. M., Park, K. S., and Kwon, Y. T. (2012) The N-end rule pathway. Annu. Rev. Biochem. 81, 261-289
44. Dougan, D. A., Micevski, D., and Truscott, K. N. (2012) The N-end rule pathway: from recognition by N-recognins to destruction by AAA+proteases. Biochim. Biophys. Acta 1823, 83-91
45. Varshavsky, A. (2008) Discovery of cellular regulation by protein degradation. J. Biol. Chem. 283, 34469-34489
46. Lee, K. E., Heo, J. E., Kim, J. M., and Hwang, C. S. (2016) N-terminal acetylation-targeted N-end rule proteolytic system: the Ac/N-end rule pathway. Mol. Cells 39, 169-178
47. Turner, G. C., Du, F., and Varshavsky, A. (2000) Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway. Nature 405, 579-583
48. Hwang, C. S., Shemorry, A., and Varshavsky, A. (2009) Two proteolytic pathways regulate DNA repair by cotargeting the Mgt1 alkylguanine transferase. Proc. Natl. Acad. Sci. U.S.A. 106, 2142-2147
49. Heck, J. W., Cheung, S. K., and Hampton, R. Y. (2010) Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1. Proc. Natl. Acad. Sci. U.S.A. 107, 1106-1111
50. Eisele, F., and Wolf, D. H. (2008) Degradation of misfolded proteins in the cytoplasm by the ubiquitin ligase Ubr1. FEBS Lett. 582, 4143- 4146
51. Bachmair, A., and Varshavsky, A. (1989) The degradation signal in a short-lived protein. Cell 56, 1019-1032
52. Inobe, T., and Matouschek, A. (2014) Paradigms of protein degradation by the proteasome. Curr. Opin. Struct. Biol. 24, 156-164
53. Tobias, J. W., Shrader, T. E., Rocap, G., and Varshavsky, A. (1991) The N-end rule in bacteria. Science 254, 1374-1377
54. Mogk, A., Schmidt, R., and Bukau, B. (2007) The N-end rule pathway of regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol. 17, 165-172
55. Rivera-Rivera, I., Román-Hernández, G., Sauer, R. T., and Baker, T. A. (2014) Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates. Proc. Natl. Acad. Sci. U.S.A. 111, E3853-E3859
56. Piatkov, K. I., Vu, T. T., Hwang, C.-S., and Varshavsky, A. (2015) Formylmethionine as a degradation signal at the N-termini of bacterial proteins. Microbial Cell 2, 376-393
57. Humbard, M. A., Surkov, S., De Donatis, G. M., Jenkins, L. M., and Maurizi, M. R. (2013) The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons. J. Biol. Chem. 288, 28913-28924
58. Graciet, E., Hu, R. G., Piatkov, K., Rhee, J. H., Schwarz, E. M., and Varshavsky, A. (2006) Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen. Proc. Natl. Acad. Sci. U.S.A. 103, 3078-3083
59. Shemorry, A., Hwang, C. S., and Varshavsky, A. (2013) Control of protein uality and stoichiometries by N-terminal acetylation and the N-end rule pathway. Mol. Cell 50, 540-551
60. Park, S. E., Kim, J. M., Seok, O. H., Cho, H., Wadas, B., Kim, S. Y., Varshavsky, A., and Hwang, C. S. (2015) Control of mammalian G protein signaling by N-terminal acetylation and the N-end rule pathway. Science 347, 1249-1252
61. Aksnes, H., Hole, K., and Arnesen, T. (2015) Molecular, cellular, and physiological significance of N-terminal acetylation. Int. Rev. Cell. Mol. Biol. 316, 267-305
62. Dörfel, M. J., and Lyon, G. J. (2015) The biological functions of Naa10: from amino-terminal acetylation to human disease. Gene 567, 103-131
63. Starheim, K. K., Gevaert, K., and Arnesen, T. (2012) Protein N-terminal acettyltransferases: when the start matters. Trends Biochem. Sci. 37, 152-161
64. Piatkov, K. I., Brower, C. S., and Varshavsky, A. (2012) The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments. Proc. Natl. Acad. Sci. U.S.A. 109, E1839-E1847
65. Piatkov, K. I., Oh, J.-H., Liu, Y., and Varshavsky, A. (2014) Calpain-generated natural protein fragments as short-lived substrates of the N-end rule pathway. Proc. Natl. Acad. Sci. U.S.A. 111, E817-E826
66. Brower, C. S., Piatkov, K. I., and Varshavsky, A. (2013) Neurodegeneration-associated protein fragments as short-lived substrates of the N-end rule pathway. Mol. Cell 50, 161-171
67. Yamano, K., and Youle, R. J. (2013) PINK1 is degraded through the N-end rule pathway. Autophagy 9, 1758-1769
68. Cha-Molstad, H., Sung, K. S., Hwang, J., Kim, K. A., Yu, J. E., Yoo, Y. D., Jang, J. M., Han, D. H., Molstad, M., Kim, J. G., Lee, Y. J., Zakrzewska, A., Kim, S. H., Kim, S. T., Kim, S. Y., et al. (2015) Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat. Cell Biol. 17, 917-929
69. Wang, H., Piatkov, K. I., Brower, C. S., and Varshavsky, A. (2009) Glutamine-specific N-terminal amidase, a component of the N-end rule pathway. Mol. Cell 34, 686- 695
70. Hwang, C. S., Shemorry, A., Auerbach, D., and Varshavsky, A. (2010) The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases. Nat. Cell Biol. 12, 1177-1185
71. Varshavsky, A. (2004) Spalog and sequelog: neutral terms for spatial and sequence similarity. Curr. Biol. 14, R181-R183
72. Hassink, G., Kikkert, M., van Voorden, S., Lee, S.-J., Spaapen, R., van Laar, T., Coleman, C. S., Bartee, E., Früh, K., Chau, V., and Wiertz, E. (2005) EB4 is a C4HC3 RING-finger containing ubiquitin ligase of the endoplasmic reticulum. Biochem. J. 388, 647- 655
73. Foresti, O., Ruggiano, A., Hannibal-Bach, H. K., Ejsing, C. S., and Carvalho, P. (2013) Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4. Elife 2, e00953
74. Xiao, Q., Zhang, F., Nacev, B. A., Liu, J. O., and Pei, D. (2010) Protein N-terminal processing: substrate specificity of Escherichia coli and human methionine aminopeptidases. Biochemistry 49, 5588-5599
75. Varland, S., Osberg, C., and Arnesen, T. (2015) N-terminal modifications of cellular proteins: the enzymes involved, their substrate specificities and biological effects. Proteomics 15, 2385-2401
76. Perrot, M., Massoni, A., and Boucherie, H. (2008) Sequence requirements for N α-terminal acetylation of yeast proteins by NatA. Yeast 25, 513-527
77. Lee, D. H., and Goldberg, A. L. (1996) Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae. J. Biol. Chem. 271, 27280-27284
78. Fleming, J. A., Lightcap, E. S., Sadis, S., Thoroddsen, V., Bulawa, C. E., and Blackman, R. K. (2002) Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341. Proc. Natl. Acad. Sci. U.S.A. 99, 1461-1466
79. Lee, S. J., Liu, T., Chattoraj, A., Zhang, S. L., Wang, L., Lee, T. M., Wang, M. M., and Borjigin, J. (2009) Posttranscriptional regulation of pineal melatonin synthesis in Octodon degus. J. Pineal Res. 47, 75-81
80. Falcón, J., Coon, S. L., Besseau, L., Cazaméa-Catalan, D., Fuentès, M., Magnanou, E., Paulin, C. H., Boeuf, G., Sauzet, S., Jørgensen, E. H., Mazan, S., Wolf, Y. I., Koonin, E. V., Steinbach, P. J., Hyodo, S., and Klein, . C. (2014) Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya. Proc. Natl. Acad. Sci. U.S.A. 111, 314-319
81. Ganguly, S., Mummaneni, P., Steinbach, P. J., Klein, D. C., and Coon, S. L. (2001) Characterization of the Saccharomyces cerevisiae homolog of the melatonin rhythm enzyme arylalkylamine N-acetyltransferase. J. Biol. Chem. 276, 47239-47247
82. Kim, J. M., and Hwang, C. S. (2014) Crosstalk between the Arg/N-end and Ac/N-end rule. Cell Cycle 13, 1366-1367
83. Slater, M., Hartzell, D., Hartnett, J., Wheeler, S., Stecha, P., and Karassina, N. (2008) Achieve the protein expression level you need with the mammalian HaloTag 7 Flexi vectors. Promega Notes 100, 16-18
84. Wu, C., and Li, S. S. (2009) CelluSpots: a reproducible means of making peptide arrays for the determination of SH2 domain binding specificity. Methods Mol. Biol. 570, 197-202
85. Komander, D., and Rape, M. (2012) The ubiquitin code. Annu. Rev. Biochem. 81, 203-229
86. Inobe, T., Fishbain, S., Prakash, S., and Matouschek, A. (2011) Defining the geometry of the two-component proteasome degron. Nat. Chem. Biol. 7, 161-167
87. van Heusden, G. P., and Steensma, H. Y. (2006) Yeast 14-3-3 proteins. Yeast 23, 159-171
88. Obsil, T., and Obsilova, V. (2011) Structural basis of 14-3-3 protein functions. Semin. Cell Dev. Biol. 22, 663-672
89. Kakiuchi, K., Yamauchi, Y., Taoka, M., Iwago, M., Fujita, T., Ito, T., Song, S. Y., Sakai, A., Isobe, T., and Ichimura, T. (2007) Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae. Biochemistry 46, 7781-7792
90. Obsilova, V., Kopecka, M., Kosek, D., Kacirova, M., Kylarova, S., Rezabkova, L., and Obsil, T. (2014) Mechanisms of the 14-3-3 protein function: regulation of protein function through conformational modulation. Physiol. Res. 63, Suppl. 1, S155-S164
91. Yang, J., Kamide, K., Kokubo, Y., Takiuchi, S., Tanaka, C., Banno, M., Miwa, Y., Yoshii, M., Horio, T., Okayama, A., Tomoike, H., Kawano, Y., Miyata, T. (2005) Genetic variations of regulator of G-protein signaling in hypertensive patients and in the general population. J. Hypertens. 23, 1497-1505
92. Matsuo, M., Coon, S. L., and Klein, D. C. (2013) RGS2 is a feedback inhibitor of melatonin production in the pineal gland. FEBS Lett. 587, 1392-1398
93. Lynch, M. (2007) The Origins of Genome Architecture, Sinauer Associates, Inc., Sunderland, MA
94. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl, K. (2010) Current Protocols in Molecular Biology, Wiley-Interscience, New York
95. Guthrie, C., and Fink, G. R. (1991) Guide to yeast genetics and molecular biology. Methods Enzymol. 194, 1-863
96. Andrews, B., Boone, C., Davis, T. N., and Fields, S. (2016) Budding Yeast: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY
97. Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87-96
98. Huang, Z., Deng, J., and Borjigin, J. (2005) A novel H28Y mutation in LEC rats leads to decreased NAT protein stability in vivo and in vitro. J. Pineal Res. 39, 84-90
99. Sikorski, R. S., and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in S. cerevisiae. Genetics 122, 19-27
100. hislain, M., Dohmen, R. J., Levy, F., and Varshavsky, A. (1996) Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae. EMBO J. 15, 4884- 4899
101. Kushnirov, V. V. (2000) Rapid and reliable protein extraction from yeast. Yeast 16, 857-860
102. werdlow, P. S., Finley, D., and Varshavsky, A. (1986) Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal. Biochem. 156, 147-153