A Function for Monoubiquitination in the Internalization of a G Protein–Coupled Receptor  Jennifer Terrell, Susan Shih, Rebecca Dunn, Linda Hicke  Molecular Cell  Volume 1, Issue 2, Pages 193-202 (January 1998) DOI: 10.1016/S1097-2765(00)80020-9

Figure 1 Lysine 48 of Ubiquitin Is Required for Cell Growth but Not for α-Factor Internalization (A) Growth curves were determined after the addition of CuSO4 to a wild-type yeast strain transformed with either CUP1-Ub (LHY420) or CUP1-UbK48R/G76A (LHY423). Cells were propagated overnight and diluted to 1 × 106 cells/ml in SD minimal medium/100 μM CuSO4. The curves are the averages of two independent experiments. (B) The same yeast strains expressing either Ub or UbK48R/G76A were induced with CuSO4 exactly as for the growth curves, harvested after 12 hr of growth, and assayed for their ability to internalize 35S-labeled α factor. The curves are the average of three independent experiments, and the standard deviation is represented by error bars. Molecular Cell 1998 1, 193-202DOI: (10.1016/S1097-2765(00)80020-9)

Figure 2 Polyubiquitin Chain Formation through a Single Lysine Residue Is Not Required for Rapid α-Factor Internalization α-factor internalization assays performed at 30°C on strains carrying a plasmid-encoded ubiquitin as their only source of ubiquitin. Curves are the averages of at least three independent experiments, and the standard deviation is represented by error bars. (A) Ub (LHY460), closed circles; UbK29R (LHY483), open squares; UbK63R (LHY485), open triangles. (B) Ub (LHY460), closed circles; UbK6R/K11R (LHY481), open squares; UbK27R (LHY482), open triangles; UbK33R (LHY484), open diamonds. Molecular Cell 1998 1, 193-202DOI: (10.1016/S1097-2765(00)80020-9)

Figure 3 A Ubiquitin Lacking All Lysine Residues Can Rescue the α-Factor Internalization Defect of a Mutant (doa4) with Low Levels of Free Ubiquitin (A) α factor was bound to doa4 strains or a congenic wild-type strain on ice, unbound pheromone was removed by centrifugation, and then internalization assays were performed at 37.5°C: DOA4 (LHY491), closed circles; doa4 (LHY492), open circles; doa4 + Ub (LHY535), open diamonds; doa4 + UbK48R (LHY534), open triangles; doa4 + Ub–no Lys (LHY520), open squares. Curves are the averages of at least three independent experiments, and the standard deviation is represented by error bars. (B) Ste2p immunoblots of extracts prepared from doa4 (LHY492), doa4 + Ub–no Lys (LHY520), and doa4 + Ub (LHY535) strains. Cells were preincubated for 5 min at 37°C, and an aliquot of each strain was withdrawn before incubation with α factor (−). α factor was then added and the strains were incubated for 8 min at 37°C before another aliquot of cells was withdrawn (+). Cell aliquots were lysed by mechanical agitation with glass beads, and the resulting extracts were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with Ste2p antiserum. The asterisk denotes a cross-reacting doublet of bands that is not related to Ste2p because it was also observed in extracts from ste2Δ cells. Hyperphosphorylated forms of the receptor are indicated by an arrow, and the mono-, di-, and triubiquitinated forms of the receptor are denoted as brackets 1, 2, and 3, respectively. Molecular Cell 1998 1, 193-202DOI: (10.1016/S1097-2765(00)80020-9)

Figure 4 α-Factor Receptor Cytoplasmic Tail Mutants Schematic representation of the wild-type receptor cytoplasmic C terminus and mutant versions of the receptor. The eight lysines within the cytoplasmic tail are indicated. Lys304, which resides near the last transmembrane domain of the protein, does not serve as a ubiquitination site. The presence of lysines within the mutant receptor constructs is denoted by closed boxes. The GGG shown in the 4KtoR-UbK48R fusion represents a 17 amino acid glycine-rich linker introduced between the receptor and ubiquitin sequences in this construct. Molecular Cell 1998 1, 193-202DOI: (10.1016/S1097-2765(00)80020-9)

Figure 5 α-Factor Receptor Variants Carrying a Single Ubiquitination Site within Their Cytoplasmic Tail Are Monoubiquitinated in Response to α-Factor Binding and Internalize Pheromone Rapidly Extracts were prepared before (−) or after (+) exposing cells expressing different mutant forms of Ste2p to α factor at 37°C. The extracts were resolved by SDS–PAGE, transferred to nitrocellulose, and immunoblotted with Ste2p antiserum. The hyperphosphorylated form of each receptor is indicated by the arrow, and the mono- and diubiquitinated forms are denoted by brackets. The mobility of molecular weight standards is indicated to the left of each blot. The relative mobilities of each species were determined from a plot of the logarithm of the molecular weight against the RF value of each band. (A) 345Stop truncated receptors expressed in end4-1 strains: 345Stop (LHY279) and K337R, 345Stop (LHY280). (B) Full-length receptors expressed in end4-1 strains: wild-type receptor (LHY538), 7KtoR+K337 (LHY563), 7KtoR (LHY541), and 8KtoR (LHY542). (C) α-factor internalization assays were performed on wild-type cells expressing different variants of the full-length α-factor receptor. Curves are the averages of at least three independent experiments, and the standard deviation is represented by error bars. Wild-type Ste2p (LHY450), closed circles; Ste2p-7KtoR (LHY393), open squares; Ste2p-7KtoR+K337 (LHY536), open triangles. Molecular Cell 1998 1, 193-202DOI: (10.1016/S1097-2765(00)80020-9)

Figure 6 Ub Fused In-Frame to a Receptor Lacking Tail Lysine Residues Mediates Rapid Internalization of the Ste2p-4KtoR-Ub Fusion Protein Cells lacking a chromosomal copy of the STE2 gene were transformed with integrating plasmids encoding Ste2p-378Stop, Ste2p-4KtoR,378Stop, Ste2p-4KtoR-UbK48R, or Ste2p-4KtoR-Ub–no Lys. The transformants were assayed for their ability to internalize α factor at 30°C. The curves are the average of three independent experiments, and the standard deviation is represented by error bars. Ste2-378Stop (LHY18), open circles; Ste2p-4KtoR,378Stop (LHY319), open triangles; Ste2p-4KtoR-UbK48R (LHY558), closed circles; Ste2p-4KtoR-Ub–no Lys (LHY588), closed squares. Molecular Cell 1998 1, 193-202DOI: (10.1016/S1097-2765(00)80020-9)