Legends to Figures and Tables in Supplementary Information
Supplementary Table 1
The aim of this project was to characterise the function of essential budding-yeast
proteins of unknown function, using the degron method. The list of such proteins can be
defined in several ways. At the start of this project we selected around 200 essential
proteins that were defined as “uncharacterised” in the Yeast Proteome Database (YPD).
We saw that about 70 of the genes encoding these proteins are, by several criteria,
unlikely to represent real essential genes: most overlap with previously characterised
genes that are known to be essential, encode polypeptides that lack homologues in other
eukaryotes, and are poorly conserved even in other budding yeast species. This left,
therefore, around 130 ‘real’ essential proteins of unknown function, of which we present
104 in this paper. In this table we summarise data regarding these 104 proteins. The
behaviour of the corresponding degron strain is given, together with details of the genes
encoding the fission yeast and human homologues.
By further examination of the information in YPD we have subsequently changed
our criteria for defining the list of essential proteins of unknown function. In a number of
cases proteins are defined as “characterised” in YPD even though their function remains
unclear (for example, they may have been found in a 2-hybrid screen without any further
experiments having been performed). A more helpful approach, in our opinion, is to
consider those essential proteins that are classified in YPD as “Biological Process
Unknown”. There are currently 181 essential proteins defined in this way (as of the 14th
April 2003). Almost one third of these proteins are unlikely to be “real” essential
proteins, for the reasons discussed above (the ORFs encoding these proteins overlap with
characterised essential genes and the proteins have no homologues in other eukaryotes
and are not always conserved in closely related budding yeasts). More than half of the
remaining essential proteins of unknown function are contained within the list of 104
analysed in this study.
Supplementary Figure 1
For each of the degron strains listed in Supplementary Table 1, we compared the ability
of cells to grow under various conditions. After growth at 25°C on YPD plates
containing 0.1mM CuSO4, we re-suspended cells in PBS and made 10-fold serial
dilutions. We then placed drops containing 50000, 5000, 500 or 50 cells on plates under
3 different conditions: YPDCu at 25°C (UBR1 OFF 25°C); YPDCu at 37°C (UBR1 OFF
37°C); YPGal 37°C (UBR1 ON 37°C). The plates were incubated for 48 hours before
examination, and growth was compared to a control expressing GAL-UBR1 but lacking a
degron-fusion.
Supplementary Figure 2
Degron-fusion proteins are degraded rapidly upon shifting cells from 24°C to 37°C.
Cells were grown at 24°C, before inducing expression of Ubr1 for 35', and transferring to
37°C for the indicated times. Examples are shown of degron-strains that are inviable at
37°C (cdc102-td and cdc105-td), or that are able to grow at 37°C (cdc101-td, ydl105w-td
and ygr278w-td). All five examples relate to proteins that are reported to be essential for
viability in budding yeast, and degradation is efficient in each case. The viability at 37°C
of strains such as cdc101-td, ydl105w-td and ygr278w-td must therefore be explained in
some other way – for example, the cell may only require very little of these proteins, or
they may not actually be essential in the W303 strain background with which we work.
Supplementary Figure 3
Three novel DNA-replication factors (Cdc101/Ydr013w, Cdc102/Yjl072c,
Cdc105/Ydr489w). Here we present a summary of the microscopic data from the
experiments described in Figure 2b, as cells were shifted from 24°C to 37°C in the
presence of Ubr1.
Supplementary Figure 4
Inactivation of Cdc102 or Cdc105 blocks the continuation of replication upon release
from hydroxyurea (HU). A control strain was grown together with the Cdc102 and
Cdc105 degron-strains at 24°C in the absence of Ubr1. Cells were synchronised in G1phase by addition of mating pheromone and then released from G1-arrest into fresh
medium containing HU. Incubation was continued for 60 minutes, until budding had
occurred in all cells and expression of Ubr1 was then induced for 35’ by transferring to
YPGalactose medium containing HU. Subsequently, the temperature was raised for 60
minutes to inactivate Cdc102 or Cdc105 and cells were released from HU-arrest at 37°C.
Samples were taken every 20 minutes, so that DNA content could be measured by flow
cytometry. Chromosome replication was completed within 40 minutes in the control
strain. In contrast, very little continued replication was detected after inactivation of
Cdc102 or Cdc105.
Supplementary Figure 5
Mitosis and the firing of a late-origin of DNA replication are blocked by HU in the
cdc102-td and cdc105-td degron-strains, just as in control cells. a) Cells from the
experiment described in Figure 4 (a) and (b) were stained with the DNA-binding dye
DAPI at the end of the HU incubation. b) Replication of the late-firing origin ARS501
was measured in G1-arrested cells and also at the end of the incubation with HU.
Supplementary Figure 6
The cassette was made by sub-cloning the kanMX selectable marker (that confers
resistance to G418) as a NotI fragment into pPW66R (Dohmen, R. J., Wu, P. &
Varshavsky, A. Science 263, 1273-6 (1994)), next to the CUP1 promoter that expresses
the heat-inducible degron-cassette. A single copy of the c-myc epitope was added at the
end of the degron, followed by a linker sequence encoding five copies of a GlycineAlanine repeat, yielding the plasmid pKL187. Further details of this plasmid will be
provided upon request.
Degron-strains were then constructed by a one-step PCR approach, as described
in “Methods”.