The fate of the
general amino acid permease (Gap1) is decided according to the quality
of the nitrogen source in the medium. On urea, a poor nitrogen source
(and ammonia in S288C), Gap1 is delivered to the plasma membrane. On
glutamate, a better nitrogen source, and on ammonia in a
lst4delta mutant, Gap1 is sent to the vacuole for
degradation. We are now seeking genes that control this sorting
decision which takes place in the Golgi. We demonstrate that
overexpression of Bul1p or Bul2p homologs, which bind the E3 ubiquitin
ligase Rsp5, reduces the cell surface activity of Gap1, an effect
suppressed by the Golgi to endosome trafficking mutant
pep12delta. Conversely, deletion of BUL1 and BUL2
causes a five fold increase in Gap1 activity at the plasma membrane as
compared to wild type. Gap1 is absent from the plasma membrane in a
lst4delta strain, but in a lst4delta bul1delta bul2delta
strain Gap1 is predominantly localised to the cell surface as
determined by sucrose gradient analysis and Gap1-GFP fluorescence
microscopy. This behaviour can not be explained by possible effects on
endocytosis, because lst4delta and end3delta lst4delta
strains exhibit similarly low levels of Gap1 activity. Thus Bul1/2
positively regulate Golgi to vacuole sorting of Gap1, and their effect
on sorting is epistatic to that of Lst4. We envisage that
ubiquitination of membrane proteins acts as a general sorting signal
for traffic to the vacuole, from both the Golgi as well as the plasma
membrane.
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