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. 2000 Apr 11;97(8):4034-9.
doi: 10.1073/pnas.070044097.

The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae

S Springer  1 , E Chen, R Duden, M Marzioch, A Rowley, S Hamamoto, S Merchant, R Schekman
Affiliations

The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae

S Springer et al. Proc Natl Acad Sci U S A. .

Abstract

To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S. cerevisiae (p24Delta8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24Delta8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single Deltaemp24 deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.

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Figures

Figure 1
Figure 1
Cells bearing p24 deletions exhibit no morphological abnormalities. Electron micrographs of logarithmic-phase cells grown in YPD (yeast extract/peptone/dextrose) at 30°C. (Bar = 1 μm.)
Figure 2
Figure 2
Cells bearing p24 deletions are secretion competent. Cells were preshifted to 37°C to induce the sec21-3 phenotype, pulse labeled for 10 min, and chased for the indicated times. Labeled proteins from the supernatant were separated by SDS–PAGE and detected by autoradiography.
Figure 3
Figure 3
The Gas1p maturation delay of Δemp24 cells is not exacerbated in p24Δ4 and p24Δ8 cells. Cells were pulse labeled for 5 min and chased for the indicated times. Gas1p was immunoprecipitated from cell extracts, resolved by SDS–PAGE, and detected by autoradiography. Migration positions of mature (125-kDa) and immature (105-kDa) forms are indicated.
Figure 4
Figure 4
The invertase maturation delay in Δemp24 cells is not exacerbated in p24Δ4 and p24Δ8 cells. Cells expressing invertase from the CPY promoter were pulse labeled for 10 min and chased for 10 min. Spheroplasts were prepared and centrifuged to obtain the cytoplasmic (I, internal) and periplasmic (E, external) fractions. Invertase was immunoprecipitated, resolved by SDS–PAGE, and detected by autoradiography. Migration positions of core-glycosylated (ER form) and hypo- and hyperglycosylated (Golgi forms) are indicated.
Figure 5
Figure 5
CPY secretion is not affected in p24 deletion strains. Cells were pulse labeled for 5 min and chased for the indicated times. CPY was immunoprecipitated from cell extracts, resolved by SDS–PAGE, and detected by autoradiography. Migration positions of p1 (ER), p2 (Golgi), and mature (vacuole) forms are indicated.
Figure 6
Figure 6
Retrieval of Suc2-Wbp1p-KKTN (KK) and Suc2-Wbp1p-QKTN (QK) in wild-type, mutant, and p24 deletion strains. Cells were pulse labeled for 10 min and chased for 0 or 1 h. The fusion protein was immunoprecipitated from cell extracts, treated with endoglycosidase H, resolved by SDS–PAGE, and detected by autoradiography. Migration positions of immature and processed (vacuole) forms are indicated. Percentages of processed forms are averages of three experiments with standard deviations where applicable. The asterisk denotes a band that may represent either an intermediate PEP4-independent proteolytic product or nonspecific cross-reactive material, which was not included in the quantitation.
Figure 7
Figure 7
The Kar2p retention defect in Δemp24 cells is not exacerbated in p24Δ4 and p24Δ8 cells. Cells were transferred to fresh media, and Kar2p was immunoprecipitated from supernatants withdrawn at the indicated times, resolved by SDS–PAGE, and detected by immunoblotting.
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