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First published online 9 March 2004
doi: 10.1242/jcs.01019

Journal of Cell Science 117, 1665-1673 (2004)
Published by The Company of Biologists 2004
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Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p

Netta Fatal1,*, Leena Karhinen1,*, Eija Jokitalo2 and Marja Makarow1,3,{ddagger}

1 Program in Cellular Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland
2 Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland
3 Department of Applied Chemistry and Microbiology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland



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  		Fig. 1. Hsp150 variants and dependence of their ER exit on functional Sec24p. (A) The product of the HSP150 gene has an N-terminal 18 amino acid signal peptide (SP, black). The ER form consists of a 54 amino acid subunit I (SU I, gray) and subunit II (SU II), which is composed of a repetitive region (RR) where homologous peptides of mostly 19 amino acids are repeated 11 times (diagonally striped boxes), followed by a unique C-terminal fragment (white area). (B) The last 89 amino acids of Hsp150 were deleted. (C) The C-terminal 114 amino acids of Hsp150 were joined to subunit I. (D) E. coli ß-lactamase (criss-crossed) was joined to subunit I. (E) HRP (checkered) was fused to the first 321 amino acids of Hsp150. (F) Invertase (horizontally striped) was fused to subunit I, and Cterm was fused to the C-terminus of invertase. (G) Invertase was fused to the C-terminus of SUI-Cterm. The last amino acids of the various domains are numbered. Letters indicate extra amino acids resulting from cloning strategy. All other proteins had a Kex2p recognition site at the C-terminal end of subunit I, except SUI-ß-lactamase (D) and Hsp150{Delta}-HRP (E). The dependency of ER exit on functional Sec24p is indicated.

 


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  		Fig. 2. Intracellular transport of proteins in sec24-1 mutant. (A) Hsp150 secretion. sec24-1 (H1101; lanes 1-10) and sec18-1 (H4, lane 11) cells were pulse-labeled for 5 minutes with 35S-methionine/cysteine and chased in the presence of CHX at 37°C as indicated. Preincubation before labeling at 37°C was for 15 minutes, except in the case of lanes 9 and 10, where it was 30 minutes. Immunoprecipitation of cell lysate (c) and medium (m) samples before SDS-PAGE analysis was with Hsp150 antiserum. Migration of ER (97 kDa) and mature (150 kDa) forms of Hsp150 are indicated on the right. (B) Transport to the vacuole of pro-CPY. sec24-1 cells (H1101) were preincubated for 15 minutes and 35S-labeled for 5 minutes and chased with CHX as indicated, at 37°C (lanes 1-4) or at 24°C (lanes 5-8). Cell lysates were immunoprecipitated with CPY antiserum. Migration of cytosolic (c, 59 kDa), ER (p1, 67 kDa), Golgi (p2, 69 kDa) and vacuolar (m, 62 kDa) forms of CPY are indicated. (C) Invertase secretion. Control (H23, lane 1), sec18-1 (H4, lane 2) and sec24-1 (H1101, lanes 3 and 4) cells were preincubated at 38°C (lanes 1-3) or 24°C (lane 4) for 15 minutes, shifted to low glucose (0.1%) medium to derepress invertase synthesis and incubated for 1 hour at the same temperatures. After nondenaturing gel electrophoresis the gel was stained for invertase activity. Migration of mature invertase is indicated. T, temperature.

 


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  		Fig. 3. Secretion of the C-terminal domain of Hsp150 in a sec24-1 mutant. sec24-1 (H1500, A), sec13-1 (H1429, B) and control cells (H1508, C) were preincubated for 15 minutes, 35S-labeled for 5 minutes and chased as indicated, at 37°C. Cell lysate samples (c) and the respective medium samples (m) were subjected to immunoprecipitation with Hsp150 antiserum and SDS-PAGE (15% gels) analysis. SUI-Cterm and released Cterm are indicated.

 


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  		Fig. 4. Fate of Hsp150{Delta} in sec24-1 mutant. sec24-1 (H1499), control (H430) and sec18-1 (H440) cells were preincubated for 15 minutes, pulse-labeled for 5 minutes and chased as indicated. The ER and mature forms of Hsp150{Delta} are indicated on the right and molecular weight markers (kDa) on the left.

 


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  		Fig. 5. Electron microscopy of Hsp150{Delta}-HRP. sec24-1 (A; H1458), sec23-1 (C; H1459) and sec7-1 (D, H1455) mutants expressing Hsp150{Delta}-HRP, and the parental sec7-1 strain (B; H3) were incubated for 1 hour at 37°C, fixed and processed for HRP EM. Large black arrows indicate putative stained ER (A and C), or Golgi cisternae (D). The large open arrow points to unidentified unstained membrane (A). Small arrows indicate stained (C) and unstained (D) nuclear membrane. CW, cell wall; G, Golgi; N, nucleus; PM, plasma membrane; V, vacuole. Bars, 0.5 µm.

 


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  		Fig. 6. The C-terminal domain of Hsp150 as mediator of ER exit of invertase. (A) Control (H1540 in lane 1, and H1577 in lane 4), sec24-1 (H1544 in lane 2, and H1579 in lane 5), sec18-1 (H1542 in lane 3, and H1575 in lane 6), and sec13-1 (H1578 in lane 7) cells were 35S-labeled for 5 minutes after a 15-minute preincubation, and chased with CHX for 40 minutes at 37°C. After immunoprecipitation with Hsp150 antiserum and endo H digestion, the samples were subjected to SDS-PAGE analysis. The recombinant strains expressed SUI-Cterm-invertase or SUI-invertase-Cterm, as indicated. The apparent molecular weights of the immunoprecipitates (kDa) are indicated on the right. (B) sec18-1 (H1542), control (H1540), sec24-1 (H1544) and sec13-1 (H1578) cells were labeled as above, but immunoprecipitated with CPY antiserum. The ER (p1, 67 kDa), mature (m, 62 kDa) and cytosolic (c, 59 kDa) forms are indicated.

 


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  		Fig. 7. Hsp150 secretion in the absence of Sfb2p. Mutants (A) sec24-1 (H1101) and (B) sec24-1 {Delta}sfb2 (H1555) were preincubated at 37°C for 15 minutes and pulse-labeled for 5 minutes with 35S-methionine/cysteine, followed by chase in the presence of CHX at 37°C as indicated. Immunoprecipitation of cell lysate (c) and medium (m) samples before SDS-PAGE analysis was with Hsp150 antiserum. Migration of marker proteins (M) is indicated on the left.

 

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© The Company of Biologists Ltd 2004