The a-factor receptor (Ste3p) is
subject to two mechanistically distinct modes of endocytosis. In the
absence of its pheromone ligand a-factor, Ste3p undergoes a
rapid, ubiquitin-dependent endocytosis which delivers surface receptor
to the vacuole for degradation. When this constitutive uptake pathway
is mutationally blocked, an a-factor-dependent uptake may be
observed. Rather than being degradatory, this ligand-dependent pathway
instead links to recycling of the internalized receptor. In our
typical experiment, exposure of cells to a-factor ligand
continuously present in the medium leads to receptor internalization
with an endpoint distribution of the receptor with approximately 70%
internalized to endosomes and 30% remaining at the surface. This
endpoint appears actually to be an equilibrium point where the ongoing
uptake is balanced by the rate of recycling return of receptor to the
surface. Indeed, following removal of ligand from the culture medium,
one observes a clear return of receptor to the surface. While this
work has predominantly utilized Ste3p mutants that lack the signal for
constitutive endocytosis signal, effects of a-factor on
wild-type Ste3p trafficking are consistent with a ligand-dependent
switch to a recycling mode. Finally, though this ligand-dependent
recycling mode of endocytosis is associated with ubiquitination,
preliminary indications are that this modification may be functioning
at a post-surface, i.e. endosomal, trafficking step.
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