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First published online 7 December 2004
doi: 10.1242/jcs.01583

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Ice2p is important for the distribution and structure of the cortical ER network in Saccharomyces cerevisiae

¹ Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA
² Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06519, USA

View larger version (49K): [in a new window]   Fig. 1. The ER fails to distribute to the periphery of daughter cells in the absence of Ice2p. Wild-type and ice2 mutant cells expressing the ER markers Hmg1p-GFP (A), or Sec61p-GFP (B) were grown at 30°C to early log phase in minimal medium with the appropriate amino acids. Asterisks indicate small buds in ice2 cells without cortical ER; arrows and arrowheads point to buds with a concentration of ER at the tip. The ER distribution in small buds with a volume of 4-13% of the mother cell was analysed using fluorescence microscopy.

View larger version (22K): [in a new window]   Fig. 2. The transposon insertion specifically affects YIL090w expression. (A) Mapping of the transposon insertion site in TMT561. Insertion of the Tn3 transposon in TMT561 (strain SFNY1052) maps between the ORFs YIL091c and YIL090w on chromosome IX. (B) The Tn3 transposon specifically affects expression of YIL090w. RT-PCR was performed on samples prepared from the wild type (lanes 2-4) and from TMT561 (lanes 5-7). Primers for YIL090w (lanes 2, 5) and YIL091c (lanes 3, 6) were used to determine whether the message levels in either ORF were affected by the Tn3 transposon. Primers for AUX1/SWA2 were used as a positive control (lanes 4, 7). (C) RT-PCR was performed on RNA samples prepared from wild type (lanes 2-5, 7) and TMT561 (lanes 6, 8). Primers for YIL090w (lanes 2-6) were used to determine message levels at increasing concentrations of RNA. Primers for AUX1/SWA2 were used as a positive control (lanes 7, 8). Lane 1 in B and C contain the molecular weight standards.

View larger version (28K): [in a new window]   Fig. 3. Predicted topology of Ice2p. (A) Ice2p might aggregate at 100°C. Lysates were prepared from strains SFNY1082 (Ice2p-GFP) and NY873 (an untagged control strain). Protein samples were TCA precipitated and subjected to SDS-PAGE and western blot analysis using an affinity-purified polyclonal antibody directed against GFP (lanes 1,2). A band corresponding to Ice2p-GFP that migrated below the 66 kDa marker was found in lane 1 but not in the untagged control (lane 2). Ice2p-GFP was not detected when samples in lanes 3 and 4 (untagged control) were heated for 5 minutes at 100°C in sample buffer. Lysates were also prepared from a strain in which Ice2p was tagged at its C-terminus with a 13x-Myc epitope. Samples were TCA precipitated, resolved on a 10% SDS-polyacrylamide gel and subjected to immunoblot analysis using monoclonal antibodies directed against the Myc epitope on Ice2p. Lane 5 contains Ice2p-Myc. Lane 6 contains a lysate prepared from an untagged strain (NY873). (B) The mobility of Ice2p was not altered upon EndoH treatment. Lysates from the untagged control and SFNY1283 (Ice2p-Myc) were digested with EndoH (lanes 2, 4) for 16 hours. Samples were resolved by SDS-PAGE and subjected to western blot analysis using anti-Myc (B, top) and anti-CPY antibodies (B, bottom). (C) Schematic of Ice2p topology. Putative glycosylation sites lie in the second and third cytosolic loops of Ice2p (asterisks). This analysis supports the hypothesis that Ice2p is a type-III membrane protein.

View larger version (13K): [in a new window]   Fig. 4. Ice2p fractionates with the ER and not the plasma membrane. Cells were grown in YPD at 30°C to early log phase. (A) Ice2p-Myc (black dashed line) fractionates with the ER but not the Golgi peak of Sec22p (grey solid line) on a sucrose velocity gradient. (B) Ice2p-Myc (black dashed line) fractionates with the ER marker Sec61p (black solid line), but does not fractionate with the plasma-membrane marker Pma1p (grey solid line) on a sucrose density gradient containing EDTA.

View larger version (56K): [in a new window]   Fig. 5. Ice2p is found on the perinuclear and cortical ER. (A) Cells expressing a GFP fusion to Ice2p were grown at 30°C in minimal medium with the appropriate amino acids to early log phase and analysed by fluorescence microscopy. (B) Ice2p localizes with the ER marker Hmg1p-GFP. Ice2p was tagged at its C-terminus with 13x-Myc in cells expressing the ER marker Hmg1p-GFP (strain SFNY1283). The distribution of Ice2p was determined by indirect immunofluorescence using anti-myc monoclonal antibody. The first row shows Hmg1p-GFP. The anti-myc immunofluorescence is shown in the second row. The third row shows the merged image. The last row represents the differential-interference-contrast image.

View larger version (48K): [in a new window]   Fig. 6. The cortical ER tubular network is defective in the ice2 mutant. Wild-type (strain SFNY1061) (A), ice2 mutant (strain SFNY1282) (B) and sec3 mutant (strain SFNY1449) (C) cells expressing the ER marker Hmg1p-GFP were analysed using fluorescence microscopy. Images were obtained from the centre of the cell and from the cell periphery. Arrowheads point to a continuous network displayed in the mother cell (A,C). Arrows point to buds that display a normal cortical ER network (A) and buds that lack an intact cortical ER network (B). An abnormal ER network is observed in the mother cell of the ice2 mutant (B). (D) Quantitation of wild-type and mutant cells displaying an aberrant cortical ER network.

View larger version (36K): [in a new window]   Fig. 7. ER membranes from ice2 mutant cells do not fractionate with the ER peak of wild-type membranes. Wild-type (strain SFNY1054) and ice2 (strain SFNY1128) mutant cells were grown at 30°C in YPD to early log phase and subjected to subcellular fractionation using sucrose velocity (A) and sucrose density gradients (B). Fractions were then subjected to western blot analysis. In the wild type, the ER markers Sec61p (A, left) and Hmg1p-GFP (A, middle), and the ER peak of Bet1p (A, right) were found in fraction 9 (black squares), whereas the ER from ice2 cells was found in fraction 7 (grey triangles). In wild-type density gradients, the ER markers Sec61p (B, left) and Aux1p (B, middle) were found in fraction 10 (black squares), whereas the ER from ice2 cells was shifted to the left in fraction 9 (grey triangles). The distribution of the Golgi was not altered in ice2 mutants (A, right, B, right). (C) ER membranes from the ice2 mutant fractionate with wild-type ER after cycloheximide treatment. The SFNY1054 and SFNY1128 strains expressing the ER marker Hmg1p-GFP were grown in YPD at 30°C and treated with cycloheximide for 6 hours, and then subcellular fractionation was performed using sucrose density gradients. Western blot analysis demonstrated that the ER markers Sec61p (C, left), Aux1p (C, middle) and Dpm1p (C, right), were in fraction 10 in both wild-type (black triangles) and ice2 samples (grey squares). (D) Cycloheximide treatment restores the density of srp102-510 ER membranes to that of the wild type. Wild-type (FY23) and srp102-510 (WPY152) mutant cells were grown at 25°C in YPD to early log phase, shifted to 37°C for 6 hours in the absence (D, left, middle) or presence (D, right) of cycloheximide (CHX) and subjected to subcellular fractionation using sucrose density gradients. Western blot analysis showed that ER membranes (D, left) from the wild type (black squares) peaked in fraction 10, whereas the ER from srp102-510 was found in fraction 9 (grey triangles). Upon CHX treatment, the ER marker Dpm1p (D, right) was in fraction 9 in both the wild type (black squares) and srp102-510 (grey triangles).

View larger version (63K): [in a new window]   Fig. 8. An aberrant cortical ER network persists after cycloheximide treatment in the ice2 mutant. (A) The cortical ER network remains abnormal upon cycloheximide treatment in the ice2 mutant. Diploid cells (strains SFNY1061 and SFNY1282) expressing the ER marker Hmg1p-GFP were grown in YPD at 30°C, treated with cycloheximide for 6 hours and analysed using fluorescence microscopy. Images were obtained from the periphery and centre of the cell. Arrowheads point to a continuous network displayed in wild-type mother cells (A, left). Arrows point to wild-type buds that display a normal cortical ER network (A, left) and to buds in ice2 cells that lack an intact cortical ER network (A, right). An abnormal ER network (arrowheads) is also observed in the mother cell of the ice2 mutant (A, right). (B) The mitochondrial structure remains intact in ice2 mutants displaying an aberrant cortical ER network. Mitochondrial distribution was studied using fluorescence microscopy in wild-type and ice2 cells expressing the ER marker Hmg1p-GFP (left). Mitochondria were visualized in cells transformed with plasmid SFNB798, which expresses the mitochondrial targeting sequence from the F0 ATP synthase fused to RFP (middle). The differential-interference-contrast images (DIC) are located on the right. The distribution of mitochondria was determined in small buds with a volume of 4-13% of the mother.