A. P. Newman

G. Rossi

J. Graf

J. P. Lian

MANCINI, Patrizia

S. Ferro Novick

English

PubMed

Archivio della ricerca- Università di Roma La Sapienza

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1992, P. 3663-36640270-7306/92/083663-02$02.00/0Letter to the EditorSEC22 and SLY2 Are IdenticalGenetic analysis of the secretory pathway in the yeastSaccharomyces cerevisiae has identified 11 sec and betmutants that are temperature sensitive for transport from theendoplasmic reticulum (ER) to the Golgi complex (secl2,-13, -16, -17, -18, -20, -21, -22, and -23 and betl and -2 [6, 8]).To clone SEC22, we transformed the sec22-3 mutant with alibrary of yeast genomic DNA constructed in the multicopyvector YEP24 (2). We defined a 4.5-kb fragment that com-plemented this mutant at 37°C. Integration by homologousrecombination was used to determine whether this DNAfragment contained the SEC22 structural gene. This wasdone by ligating the 4.5-kb insert into YIp5, a URA3-containing plasmid that must be integrated into the yeastgenome to be maintained (12). The resulting construct wasdigested within the insert to direct integration and thentransformed into a ura3 his4 strain. These events placed theURA3 gene adjacent to the cloned locus. The Ura+ strainwas then crossed to a ura3 sec22-3 mutant. The diploid wassporulated, and the Ts' and Ura+ markers were found tocosegregate in 15 of 15 tetrads. This finding indicates that thecloned DNA contains the SEC22 structural gene.Further subcloning defined a 2.8-kb fragment and a 1.8-kbfragment (Fig. 1A and B) capable of full complementation.Digestion of the unique NcoI site followed by end filling andligation resulted in a loss of complementing activity, definingthis site as internal to the gene (Fig. 1E). DNA sequenceanalysis identified one open reading frame (of 0.64 kb)spanning this site, which is predicted to encode a 25-kDaprotein. A comparison of this sequence with others in theNational Biomedical Research Foundation Library data baserevealed that SEC22 is identical to SLY2 (suppressor of lossof YPT1 function). SLY2 was identified as a gene whoseoverproduction enables yeast cells to survive in the absenceof the essential ras-like Yptl protein (3, 4), which is requiredfor ER-to-Golgi transport (1, 10, 11).SEC22 interacts genetically with BET] and BOSJ, twoother genes required for ER-to-Golgi transport (7). To iden-tify additional genetic interactions, one member of eachcomplementation group of sec and bet mutants that blockER-to-Golgi transport was transformed with the SEC22 geneon a multicopy plasmid. The sec22-3 mutant defect was fullyrescued, as expected, and sec21-1 was suppressed (5). Thesec21-1 mutant grows at 30°C but not at 34°C. However, inthe presence of the multicopy plasmid, the transformedsec2l strain displays moderate growth at 34°C but is notrescued at 37°C. SLY2 was found to suppress sec21-1 over asimilar temperature range (9). Only partial suppression ofsec22-3 by SLY2 was reported (9). The reason for this isunclear, since a BamHI-EcoRV fragment was used, which ispresumably identical to the fully complementing plasmidpSFNB50 (Fig. 1D). However, the integration experimentand the DNA sequence identity unequivocally demonstratethat SEC22 and SLY2 are the same gene. The identity ofSEC22 and SLY2 is further supported by the observationthat antisera raised to either a lacZ-SLY2 (9) or a GST-SEC22 fusion protein recognize a band of 25 kDa thatpartitions with membranes.We thank Melissa Clague and Caryn Hashimoto for technicalassistance. We also thank Irene Griff for the comparison of SEC22with other sequences in the data base.This work was supported by grants awarded to S.F-N from theNational Institutes of Health (1 R01 GM45431 and CA 46128).REFERENCES1. Bacon, R. A., A. Salminen, H. Ruohola, P. Novick, and S.Ferro-Novick. 1989. The GTP-binding protein, Yptl, is requiredfor transport in vitro: the Golgi apparatus is defective in yptlmutants. J. Cell Biol. 109:1015-1022.2. Carlson, M., and D. Botstein. 1982. Two differentially regulatedmRNAs with different 5' ends encode secreted and intracellularforms of yeast invertase. Cell 28:145-154.3. Dascher, C., R. Ossig, D. Gallwitz, and H. D. Schmitt. 1991.Identification and structure of four yeast genes (SLY) that areable to suppress the functional loss of YPTI, a member of theRAS superfamily. Mol. Cell. Biol. 11:872-885.4. Gallwitz, D., C. Donath, and C. Sander. 1983. A yeast geneencoding a protein homologous to the human c-haslbas proto-oncogene product. Nature (London) 306:704-707.5. Newman, A. P. 1991. The role of the yeast BET and BOS genesin transport between the ER and the Golgi complex. Ph.D.thesis, Yale University, New Haven, Conn.6. Newman, A. P., and S. Ferro-Novick. 1987. Characterization ofnew mutants in the early part of the yeast secretory pathwayisolated by a 3H-mannose suicide selection. J. Cell Biol. 105:1587-1594.7. Newman, A. P., J. Shim, and S. Ferro-Novick. 1990. BETI,BOSI, and SEC22 are members of a group of interacting yeastgenes required for transport from the endoplasmic reticulum tothe Golgi complex. Mol. Cell. Biol. 10:3405-3414.8. Novick, P., C. Fields, and R. Schekman. 1980. Identification of23 complementation groups required for post-translationalevents in the yeast secretory pathway. Cell 21:205-215.9. Ossig, R., C. Dascher, H.-H. Trepte, H. D. Schmitt, and D.Gallwitz. 1991. The yeast SLY gene products, suppressors ofdefects in the essential GTP-binding Yptl protein, may act inendoplasmic reticulum-to-Golgi transport. Mol. Cell. Biol. 11:2980-2993.10. Schmitt, H. D., M. Puzicha, and D. Gallwitz. 1988. Study of atemperature-sensitive mutant of the ras-related YPTI geneproduct in yeast suggests a role in the regulation of intracellularcalcium. Cell 53:635-647.11. Segev, N., J. Mulholland, and D. Botstein. 1988. The yeastGTP-binding Yptl protein and a mammalian counterpart areassociated with the secretion machinery. Cell 52:915-924.12. Struhl, K., D. Stinchcomb, S. Scherer, and R. Davis. 1979. Highfrequency transformation of yeast: autonomous replication ofhybrid DNA molecules. Proc. Natl. Acad. Sci. USA 76:1035-1039.Anna P. NewmanJennifer GrafPatrizia ManciniGuendalina RossiJian P. LianSusan Ferro-NovickDepartment of Cell BiologyYale University School ofMedicine333 Cedar StreetNew Haven, Connecticut 065103663Vol. 12, No. 8 at DIP.MEDICINA INTERNA on March 25, 2009 mcb.asm.orgDownloaded from 3664 LETTER TO THE EDITORcomplementationof sec22-3BamH PstIA. \I0.0HinclINcoI'I1.0EcoRV EcoRI EcoRVHinc]II I (2.0BamHI2.8kbB. L++C.D. L +E. IFIG. 1. Complementing activity of plasmids containing the SEC22 gene. Only the cloned insert, and not the vector portion of each plasmid,is shown. (A) pJG103; (B) pSFNB30a; (C) pSFNB30b; (D) pSFNB50; (E) pSFNB82. pJG103 was constructed in the multicopy vector YEp24,and the others were constructed in the single-copy vector YCp5O. The arrow in pJG103 indicates the direction and length of the SEC22 codingsequence, and the triangle in pSFNB82 indicates the location of the frameshift mutation.Author's ReplyIn our previous reports on the cloning and functionalanalysis of yeast genes that are capable of suppressingyptlloss-of-function mutants, we had shown that SLY2, SLY12,and SLY1-20 could partially suppress a temperature-sensi-tive, secretion-defective sec22 mutant (1, 2). After we wereinformed that SLY2 and SEC22 might be identical, we testedthe genotype of the sec22 mutant strain that we had receivedfrom another laboratory and had used in our earlier comple-mentation studies (2). We discovered that this strain, andthose derived from it, carried a Ts- allele of sec2l ratherthan sec22. We therefore reinvestigated the complementingactivities of the different SLY genes (1, 2), using a sec22-3mutant provided by R. Schekman. In agreement with theresults reported above by Newman et al., the cloned SLY2gene on a multicopy vector perfectly rescued the mutantgrowth defect at 37°C. In addition, diploid strains that wereobtained by crossing the sec22-3 mutant with a strain carry-ing the disrupted SLY2 allele were still Ts-, which alsoagrees with the above finding that SLY2 and SEC22 are infact allelic.In contrast to our previously published experiments inwhich we unknowingly used the wrong yeast strain, we nowfind that SLY1-20, a single-copy suppressor of YPTI disrup-tions (1, 2), as well as the multicopy suppressor SLY12 (1, 2)allows the sec22-3 mutant to grow at 36°C. However, SLY41(1, 2) did not improve the growth of this mutant at temper-atures higher than 30°C. This shows that SLYJ-20 and SLY12are in fact more potent suppressors of sec22 than of sec2lmutants.REFERENCES1. Dascher, C., R. Ossig, D. Gallwitz, and H. D. Schmitt. 1991.Identification and structure of four yeast genes (SLY) that areable to suppress the functional loss of YPT1, a member of theRAS superfamily. Mol. Cell. Biol. 11:872-885.2. Ossig, R., C. Dascher, H.-H. Trepte, H. D. Schmitt, and D.Gallwitz. 1991. The yeast SLY gene products, suppressors ofdefects in the essential GTP-binding Yptl protein, may act inendoplasmic reticulum-to-Golgi transport. Mol. Cell. Biol. 11:2980-2993.Rainer OssigChristiane DascherHans Dieter SchmittDieter GallwitzDepartment of Molecular GeneticsMax Planck Institute for Biophysical ChemistryPO Box 2841D-3400 Gottingen, GermanyMOL. CELL. BIOL.i at DIP.MEDICINA INTERNA on March 25, 2009 mcb.asm.orgDownloaded from 

SEC22 AND SLY2 ARE IDENTICAL

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SEC22 AND SLY2 ARE IDENTICAL

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