1. Slow Diffusion of Proteins in the Yeast Plasma Membrane Allows Polarity to Be Maintained by Endocytic Cycling 
Javier Valdez-Taubas, Hugh R.B. Pelham 
Current Biology 
Volume 13, Issue 18, Pages (September 2003)
DOI: /j.cub

2. Figure 1 Endocytosis Is Required for Snc1 Polarization
(A) Confocal microscope images of GFP-Snc1 or the en− derivative in wild-type cells, or where indicated, in end4-1 cells, with or without α factor treatment to induce shmoo formation. Fluorescence and DIC images are shown.
(B) Expression of Snc1 from a GAL promoter was repressed for 40 min, α factor was added, and cells were imaged 2 hr later. Inverted images of long shmoos are also shown. Note the depletion of Snc1 en− from the tip.
Current Biology  , DOI: ( /j.cub )

3. Figure 2 Mobility of GFP-Tagged Proteins Measured by FRAP
Each graph contains several independent measurements of bleached areas (all 1.6 μm in diameter) represented by different symbols and normalized so that pre-bleach intensity is 1 and the intensity immediately post-bleach is 0. The curved line was manually fitted to the data for Sso1 in wild-type yeast cells and was repeated in each graph for comparison. Note the different time scales for the COS cell and vacuole data. Example images are shown at the bottom. The arrows indicate bleached areas; the white scale bars are 5 μm. For the shmoo experiment, the graph shows data from a single shmoo, together with its pre-bleach image; the areas bleached are indicated by circles. From these data, approximate diffusion coefficients were calculated as follows (μm2/s): Sso1 in COS cells, 0.103; Sso1 in yeast, ; Snc1 in yeast, ; PNTS on vacuolar membrane, 0.068; Snc1 in shmoos, ; Snc1 in spheroplasts, , Snc1 in latrunculin-treated cells, ; Snc1 in erg6 cells,
Current Biology  , DOI: ( /j.cub )

4. Figure 3
Endocytic Cycling Is Necessary and Sufficient for Polarization
(A) The addition of an endocytic signal is sufficient to polarize Sso1. GFP-Sso1 is not polarized in budding cells or shmoos, whereas NPF-GFP-Sso1 concentrates at sites of exocytosis, namely, small buds, the site of septum formation, and shmoo tips.
(B) The polarized distribution of YBR016w involves endocytosis. A GFP-tagged chimera containing residues 1–106 of YBR016w fused to the Sso1 transmembrane domain reaches the cell surface in end4 cells, but in ric1 cells, in which recycling of endocytosed proteins to the Golgi is blocked, it is on vacuoles (which are characteristically fragmented in this mutant). These findings indicate the presence of an endocytic signal. GFP-YBR016w itself is polarized in wild-type cells, but not in an end4 mutant.
Current Biology  , DOI: ( /j.cub )

5. Figure 4 GFP-GPI and Filipin Are Not Reliable Raft Markers in Yeast
(A) GFP-GPI in wild-type cells. Note the enrichment at the bud necks and scars, as observed with other GPI-linked proteins.
(B) FRAP measurements as in Figure 2 show that most of the GFP-GPI is completely immobile. Rapid recovery in the first few seconds may represent a minor, highly-mobile fraction.
(C) GFP-GPI synthesis was repressed by the addition of glucose before exposure of cells to α factor. Note the absence of fluorescence from the newly grown shmoo tips and the sharp boundary with the rest of the cell (arrows).
(D) Live cells incubated with filipin and then with FM4-64, and imaged for each, show colocalization of the two reagents in a nascent bud.
(E) Shmoos fixed with formaldehyde before incubation with filipin. Note the even staining of the plasma membrane.
Current Biology  , DOI: ( /j.cub )