Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum - PubMed (original) (raw)

Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum

K Plath et al. J Cell Biol. 2000.

Abstract

In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.

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Figures

Figure 1

Figure 1

Ribosome-bound ppαF interacts mainly with SRP54 and NAC. Fragments of 160 amino acids of K5, K5Δ, wt, and M2 ppαF containing photoreactive lysine derivatives were synthesized in the reticulocyte lysate system. RNCs were isolated, irradiated with UV light as indicated, and analyzed by SDS-PAGE and autoradiography. Non-cross-linked ppαF and cross-linked products of ppαF to SRP54 (*) and the α (○) and β subunits (•) of NAC are indicated.

Figure 2

Figure 2

SRP and NAC only interact with RNCs. A fragment of 160 amino acids of ppαF containing a single photoreactive lysine derivative at position 10 (K10 ppαF) was synthesized in vitro. RNCs were isolated, and after addition of ribosome-depleted reticulocyte lysate were incubated with or without EDTA. Full-length (f.l.) ppαF was synthesized in parallel and incubated with or without EDTA. After irradiation, the total products (totals) and products immunoprecipitated with antibodies to SRP54 or NACβ after denaturation in SDS (IP) were analyzed by SDS-PAGE. Control IPs were done without antibodies (−). The starting material for IPs was 3.5 that for the totals. Cross-linked products of ppαF to SRP54 (*) and to the α (○) and β subunits (•) of NAC are indicated. Cross-linked products of ppαF to Hsp70 (▪) and TCP1α (□) are also shown. The band marked with a triangle (▴) is an artefact of the gel.

Figure 3

Figure 3

Systematic probing of the molecular environment of full-length ppαF. Full-length polypeptide chains of wt ppαF, of ppαF molecules that each contain a single lysine at the indicated positions (pos.), and of the signal sequence mutants M2 ppαF and K5Δ ppαF were synthesized in vitro in the presence of modified lysyl tRNA. The samples were irradiated with UV light (UV) as indicated and subjected to SDS-PAGE and autoradiography. The position of non-cross-linked ppαF is indicated. Major cross-linked products are indicated by the approximate molecular weight of the cross-linked cytosolic protein. The arrow points to a product originating from internal cross-linking within ppαF. Most bands that are not dependent on irradiation correspond to ubiquitinated ppαF (*) (data not shown). Note that non-cross-linked ppαF runs somewhat differently depending on the position of the lysine codon (in particular, positions 27–43).

Figure 4

Figure 4

Full-length ppαF interacts with Hsp70 and TCP1α. (A) ppαF containing a photoreactive lysine derivative at position 10 of the signal sequence (K10 ppαF) was synthesized in vitro and irradiated with UV light (UV) as indicated. The samples were either analyzed directly by SDS-PAGE and autoradiography (total), or first immunoprecipitated (IP) with antibodies to Hsp70 and TCP1α before analysis by SDS-PAGE. IPs were performed either under native conditions or after denaturation with SDS. Control IPs were done without antibodies (−). Starting material for the IPs was 10 times that for the totals. Cross-linked products of ppαF are indicated as in the legend to Fig. 3. Note that some non-cross-linked ppαF and ubiquitinated ppαF were coprecipitated in a nonspecific manner, particularly under native conditions. (B) As in A, with ppαF containing a probe at position 97. (C) As in A, with wt ppαF. IPs were performed after denaturation in SDS. (D) As in C, with the signal sequence mutant M2 ppαF. (E) As in A, with proOmpA.

Figure 5

Figure 5

Hsp70 and TRiC/CCT form different complexes with ppαF. (A) K10 ppαF was synthesized in vitro and irradiated with UV light (UV) as indicated (total). An aliquot of the irradiated sample was subjected to sucrose gradient centrifugation, and fractions were collected from the top (lane 1) to the bottom (lane 12). Cross-linked products of ppαF are indicated as in the legend to Fig. 3. Molecular markers were run in parallel sucrose gradients, and their molecular weights are indicated. (B) As in A, with proOmpA.

Figure 6

Figure 6

All ppαF populations are translocation competent. Wt ppαF without photoreactive lysine derivatives was synthesized in vitro and subjected to sucrose gradient centrifugation. The top panel shows the distribution of radiolabeled ppαF in the different fractions in two experiments (exp1 and exp2). Translocation of ppαF in the various fractions and in the starting material (control) was tested with proteoliposomes containing Sec complex, Kar2p, and ATP. Either equal volumes or equal amounts of radioactive ppαF were analyzed. % transport, the amount of ppαF translocated relative to the total amount of ppαF in the reaction. Translocation in the absence of proteoliposomes was not observed (data not shown). The increasing amounts of sucrose in the gradient fractions had no influence on the translocation efficiency of ppαF (data not shown).

Figure 7

Figure 7

Dissociation of cytosolic complexes of ppαF. (A) K11 ppαF was synthesized in reticulocyte lysate and incubated at 30°C either without additions (♦, control), or with a GroEL trap (▵), or with proteoliposomes containing Sec complex (×). At different time points, equal aliquots were removed from the samples, irradiated with UV light, and analyzed by SDS-PAGE. The cross-linked product containing the 50-kD protein (p50) was quantitated with a PhosphorImager. The yield of cross-linked product is expressed relative to the amount of ppαF contained in the sample. The first time point was taken after 0.5 min, which already led to some binding of ppαF to the Sec complex or GroEL (see D) and to less cross-linking to cytosolic proteins compared with the control. (B) As in A for Hsp70 (p70). (C) As in A for the p60 subunit of the TRiC complex. (D) Quantitation of the ppαF cross-links to Sec62 and Sec71p (for the sample incubated with Sec complex) and to GroEL (for the sample incubated with the GroEL trap). (E) K11 ppαF was synthesized in reticulocyte lysate and incubated for 30 min at 30°C or 0°C without additions as indicated (preinc.). Half of the sample was irradiated with UV light and analyzed by SDS-PAGE. The cross-linked products containing the 50-kD protein (p50) and the TRiC subunit p60 were quantitated with a PhosphorImager. The other half of the sample was analyzed for binding of ppαF to Sec complex. The sample was incubated with proteoliposomes containing Sec complex, solubilized in digitonin, and subjected to IP for the Sec complex. ppαF coimmunoprecipitated was quantitated. The yield of cross-linked products and the efficiency of Sec complex binding are given relative to results obtained with a sample that was immediately subjected to cross-linking and binding without preincubation (control = 100%).

Figure 8

Figure 8

ppαF bound to Sec complex is not associated with cytosolic proteins. (A) K11 ppαF was synthesized in vitro. Some samples were first irradiated with UV light and then incubated with reconstituted proteoliposomes containing Sec complex (1.X/2.B). Other samples were first incubated with proteoliposomes and then irradiated with UV light (1.B/2.X). One part of each sample was analyzed directly by SDS-PAGE and autoradiography (total). Seven parts of the irradiated samples were treated with digitonin to solubilize the vesicles, and ppαF bound or cross-linked to components of the Sec complex was coimmunoprecipitated with the Sec complex (IP). Cytosolic interaction partners of ppαF are indicated as in the legend to Fig. 3. Cross-links to the Sec proteins are marked. (B) As in A, with K13 ppαF. (C) As in A, with wt ppαF. (D) As in A, with K117 ppαF. (E) As in A, with K138 ppαF.

Figure 9

Figure 9

Pathway of a posttranslational translocation substrate from the ribosome to the Sec complex. For explanation, see text.

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