Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Mar 28;97(7):3254-9.
doi: 10.1073/pnas.97.7.3254.

Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast

M Bagnat  1 , S Keränen, A Shevchenko, A Shevchenko, K Simons
Affiliations

Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast

M Bagnat et al. Proc Natl Acad Sci U S A. .

Abstract

Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Isolation of DIGs from yeast cells. (a) DIGs were enriched as described in Materials and Methods. Fractions of equal volume were collected from the top of the second gradient, TCA-precipitated, and analyzed by SDS/PAGE (7.5%, upper gel; 15%, lower gel) with silver staining. Proteins peaking at the top fractions were excised and sequenced by mass spectrometry. (b) Western blot analysis of Gas1p and Pma1p distribution in the first and second density gradients. (c) GPI-anchored proteins were labeled with [3H]myoinositol (75 μCi) to steady-state. The cleared lysate was incubated with TX100 (1%) and adjusted to 40% Optiprep. The sample then was overlaid with a step gradient (30%, 0%) and centrifuged for 2 h at 55,000 (TLS55 rotor). Fractions collected from the top were TCA-precipitated and proteins were separated by SDS/PAGE (12.5%) and detected by autoradiography. The arrows indicate the position of DIG-associated protein bands.
Figure 2
Figure 2
TX100 solubility of total lipids in wild type (W303). Total floated membranes were incubated with TX100 or TNE buffer (c), adjusted to 40% Optiprep, and centrifuged in a density gradient as in Fig. 1. Lipids were extracted from the top fraction and analyzed by TLC. Labeled lipids were detected by autoradiography and quantified by phosphorimaging. PE, phosphatidylethanolamine; #, not assigned; PC, phosphatidylcholine; GL, glycolipids; PS, phosphatidylserine; IPC, inositol phosphorylceramide; MIPC, mannose-inositol-phosphorylceramide; M(IP)2C, mannose-(inositol phosphorus)2-ceramide.
Figure 3
Figure 3
DIG association in sterol-depleted membranes. erg12–1 cells (GL7) were pregrown in medium supplemented with cholesterol. Total membranes were isolated by floatation and incubated with TNE buffer or TNE containing increasing concentrations of CD (0–20 mM) for 15 min at 37°C. Gas1p raft association was analyzed by Western blotting as in Fig. 1b.
Figure 4
Figure 4
DIG association in SL depleted cells. (a) Wild-type (RH690–15D) and lcb1–100 cells (RH3804) were grown in rich medium at 24°C. The cultures were split and incubated for 2 h at 24°C or 37°C. DIG association was analyzed by Western blotting after TX100 treatment and density gradient centrifugation (fraction 1, Upper; fraction 6, Lower). (b) Wild-type (RH690–15D) and lcb1–100 cells (RH3804) were labeled with [14C]acetate for 2 h (in NaPi + 1% glucose) at 24°C or 37°C. Ergosterol insolubility was determined by TLC analysis as in Fig. 2 (n = 3). (c) Secretion of acid phosphatase to the medium in spheroplasted lcb1–100 cells incubated at 24°C or 37°C (n = 4), expressed in arbitrary units.
Figure 5
Figure 5
DIG association of Gas1p during biosynthetic transport. Cells [wild type (NY179) and sec18–1 (H891)] were preincubated at 24°C or 37°C for 5 min, pulse-labeled with [35S]methionine for 5 min, and then chased for 0 and 45 min. The samples from the two time points were taken and subjected to TX100 extraction and density gradient centrifugation. Gas1p was immunoprecipitated from fractions 1 [insoluble (I)] and 4 [soluble (S)]; the mature (m) and precursor (p) forms are indicated.
Figure 6
Figure 6
DIG association of vacuolar, ER, and plasma membrane proteins. (a) DIG association of Gas1p, V-H+ATPase (100-kDa subunit), V-ALP, and Sec61p in wild-type (W303) cells was analyzed as in Fig. 1 by Western blotting with specific antibodies. (b) hTfR was expressed in wild-type (W303) cell membranes, and DIG association was analyzed by floatation in density gradients as in Fig. 1 with or without TX100 treatment.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Simons K, van Meer G. Biochemistry. 1988;27:6197–6202. - PubMed
    1. Simons K, Ikonen E. Nature (London) 1997;387:569–572. - PubMed
    1. Brown D A, London E. J Membr Biol. 1998;164:103–114. - PubMed
    1. Brown D A, Rose J K. Cell. 1992;68:533–544. - PubMed
    1. Scheiffele P, Roth M G, Simons K. EMBO J. 1997;16:5501–5508. - PMC - PubMed

Publication types