Western blot showing expression of mutant Sec63p. The strains used in Figure 7 were treated

Transcription

1 Supplemntary figure legend Western blot showing expression of mutant Sec63p. The strains used in Figure 7 were treated with methionine to repress transcription of genomic SEC63, and total protein extracts were made. Samples were resolved on a 7.5% gel and western blotting was performed using anti- Sec63p antibodies. The positions of wild-type Sec63p, Sec63 brlp and Sec63 Jp are indicated.

2 Supplementary Methods Strain construction To create the Sec71p-3HA-expressing yeast strain MPSY18, the 3HA-encoding sequence was amplified by PCR from the pfa6a-3ha-kanmx6 plasmid, using Sec71F2 and Sec71R1, and transformed into the wild-type strain W303 by homologous recombination (39). To create the P MET -SEC63/ssh1 strain MPSY22, BYY5 was crossed to the ssh1 /MatA strain BWY465, and the resulting diploid (MPSY21) was sporulated and dissected at 24 C. The resulting tetrads were analysed for the Trp+ phenotype diagnostic of ssh1 and for failure to grow on solid medium supplemented with methionine (indicating that Sec63p had been depleted). To generate the ssh1 /sbh1 strain MPSY24, BWY465 was crossed to BWY594 and the resulting diploid (MPSY23) was sporulated and dissected at 24 C. The resulting tetrads were analysed for the Trp+ phenotype diagnostic of ssh1 and for the Ura+ phenotype diagnostic of sbh1. To generate the ssh1 /sbh1 strain MPSY26, BWY465 was crossed to BWY596 and the resulting diploid (MPSY25) was sporulated and dissected at 24 C. The resulting tetrads were analysed for the Trp+ phenotype diagnostic of ssh1 and for the G418-resistance phenotype diagnostic of sbh2. Plasmid construction The SEC71-3HA coding sequence from MPSY18 was amplified by PCR, using oligonucleotides SacISec71F and NotISec71R. The resulting PCR product was digested with SalI/NotI and inserted into prs316 (41) to generate pmps10. To enable transformation of SEC71-expressing plasmid into yeast containing URA3 plasmids pjkr2, paj7 or paj8, expressing SEC63, sec63 brl or sec63 J respectively (14, 26), the SalI/NotI fragment from

3 pmps10 was inserted into prs313 (41) to generate pmps36. For in vitro expression of Sec71p-HA, the coding region was excised from pmps10 using HindIII and inserted into pgem3 (Promega) so that it was downstream of the SP6 promoter, to generate plasmid pmps32. Plasmid pmps41, carrying the truncated version of Sec71p-HA, was created by inserting a second PacI restriction site into the coding region using 71PacIMutF and 71PacMutR, which also inserted two extra methionine codons to enhance labelling efficiency. pmps41 was then digested using PacI, and re-ligated, resulting in the removal of 432bp of coding sequence to create pmps42. To enable disulphide crosslinking, a cysteine residue was mutated into pmps42 at position 27 using 71CMut1F and 71CMut1R (pmps46). For construction of the DHC- F-encoding plasmids, a cysteine residue was mutated into amino acid residue 20 of pjd96 using DHCmutC20F and DHCmutC20R to enable chemical crosslinking (pmps53).

4 Table S1. Saccharomyces cerevisiae strains Strain Genotype Source W303 MAT ade2 his3 leu2 ura3 trp1 can1 (43) BWY500 MAT sec65-1 ade2 his3 leu2 ura3 trp1 can1 (17) BYY5 MAT ade2 his3 leu2 ura3 trp1 can1 kanmx4- P MET3 -SEC63 (19) BWY464 MAT ade2 his3 leu2 ura3 can1 ssh1::trp1 (17) BWY465 MATa ade2 his3 leu2 ura3 can1 ssh1::trp1 CMY8 MATa ade2 his3 leu2 trp1 ura3 sec61::his3 sss1::kanmx4 [psec61nocys] [psss1tmccc] BWY596 MAT ade2 his3 leu2 trp1 can1 sbh1::ura3 BWY598 MPSY18 MPSY21 MAT ade2 his3 leu2 trp1 ura3 can1 sbh2::kanmx4 MAT ade2 his3 leu2 ura3 trp1 can1 SEC71-3HAkanMX4 MATa/MAT ade2/ade2 his3/his3 leu2/leu2 ura3/ura3 trp1/ ssh1::trp1 can1/can1 SEC63/kanMX4-P MET3 -SEC63 MPSY22 MAT ade2 his3 leu2 ura3 can1 kanmx4-p MET3 - SEC63 ssh1::trp1 MPSY23 MPSY24 MPSY25 MPSY26 MATa/MAT ade2/ade2 his3/his3 leu2/leu2 ura3/ sbh1::ura3 trp1/ ssh1::trp1 can1/can1 MAT ade2 his3 leu2 can1 sbh1::ura3 ssh1::trp1 MATa/MAT ade2/ade2 his3/his3 leu2/leu2 ura3/ura3 trp1/ ssh1::trp1 can1/can1 SBH2/ sbh2::kanmx4 MAT ade2 his3 leu2 ura3 can1 sbh2::kanmx4 ssh1::trp1

5 Table S2. Oligonucleotides used in this study Strain Genotype Source af92r SacISec71F NotISec71R 71PacIMutF 71PacMutR 71CMut1F 71CMut1R DHCmutC20F DHCmutC20R Sec71F2 Sec71R1 CGCATAGTCAGGAACGTCGTATGGGTAC ATTTCAGCCTCTCTTTTATCCAAAGATAC CCGAGCTCTCTGAAATTCAACGTGGTCA ACCATTC AAGGAAAAAAGCGGCCGCAGAAAGGAG AACCCTTAGGTCTACAAC TGAGCAACCATCCATGATGTTAATTAAC GATGCCCATGATCTGTATTTC TCATGGGCATCGTTAATTAACATCATGG ATGGTTGCTCACTAATTTTTTTGGC TGGAGACGAAATGCATCTCCGTTTATAC CCCACTC ATAAACGGAGATGCATTTCGTCTCCACA ATTGGCTC TGTTGTTGAAGTGTATAGAAACGGCACA AATTCCGGC GTGCCGTTTCTATACACTTCAACAACAA AACAGTAC GAGTGGGAGCTGAAAATAAATAATGAT GGAAGATTAGTCAATCGGATCCCCGGGT TAATTAAC AACACTGAACGAGCGAATACATATCTTT GCACACAGTAGGCAGAATTCGAGCTCGT TTAAAC

6 Supplementary Figure 1 Plasmid: Methionine: SEC sec63 brl sec63 J Sec63p Sec63 brlp Sec63 Jp 50 -