APRENDIZAJE, CULTURA VIAL Y HABILIDADES SOCIALES

YA YA/LLAA30YA/LLAA32

YA/LLAA28S2YA/LLAA28S4YA/LLAA28S9

WT-K9SWT-K27S ∆

CT1 _∆CT5

∆CT29

LLAA5 LLAA13 LLAA24 YA29 YA11S5 YA16S3 YA19S2

cell lines

FIG. 30. Internalization of 49K from the cell surface is mainly directed by the LL motif in the cytoplasmic tail. Constructs for the expression of mutant proteins were based on the pSG5-49K plasmid. DNA sequences containing mutations were generated by PCR (sense: 49KUSER546; antisense: 49KdelCT, 49KYLLAAA, 49KYA, 49KLLAA) and cloned into pSG5-49K using BglII restriction sites. The mutated sequences were confirmed by sequencing. A549 cell lines stably expressing 49K WT and mutant proteins with one or both of the YXXΦ and LL motifs in the cytoplasmic tail mutated to AXXΦ and AA or with the entire cytoplasmic tail deleted (∆CT) were generated as described in Materials and Methods and analyzed by flow cytometry. Cell surface and internal 49K levels were measured and the ratio of the mean fluorescence values of cell surface and internal expression was determined for several isolated clones of 49K WT and mutants as indicated. Mean values were calculated from 4-6 independent experiments. Error bars indicate the standard error determined with SigmaPlot for Windows Version 5.00.

The ratio was determined for 2-5 clones of WT and mutant 49K proteins to exclude clonal differences. For WT 49K this ratio was 0.24-0.29. The YA/LLAA and ∆CT mutants showed clearly enlarged ratios of 0.64-0.97 and 0.51-0.68 respectively. In the LLAA mutants the ratio was slightly increased to 0.36-0.53. The YA mutants exhibited ratios of 0.19-0.28, which was comparable to WT. Thus, YA/LLAA and ∆CT, and to a lesser extent also the LLAA mutant 49K proteins were expressed in higher amounts on the cell surface than WT and YA mutant 49K proteins indicating impaired internalization.

The immunoprecipitation data demonstrated that 49K is proteolytically processed. The site of that cleavage was unknown but it had to be considered that the cleavage might occur at the cell surface. In that case the proteolytic cleavage would limit the accumulation of 49K mutants deficient in endocytosis on the cell surface. Consistent with this hypothesis, WT and mutated 49K proteins were rapidly lost from the cell surface, when cells were treated with brefeldin A to inhibit the transport of newly synthesized protein to the cell surface (Fig. 31).

Mean Fluorescence 0 10 20 30 40 50 - BFA + BFA WT-K9S YA/LLAA30 LLAA5 ∆CT5 YA11S5 WT-K27S YA/LLAA28S4 LLAA24 YA19S2 ∆CT29

FIG. 31. Rapid decrease of cell surface expression of 49K in cell lines expressing WT and mutated 49K proteins after brefeldin A treatment. Mean fluorescence of cell surface expression of 49K was determined by flow cytometry after mock (black bars) or 4 h brefeldin A treatment (5 µg/ml, grey bars) of cell lines expressing WT and mutant 49K proteins as indicated. Mean values were calculated from 4-6 independent experiments. Error bars indicate the standard error determined with SigmaPlot for Windows Version 5.00.

After 4 h BFA treatment only 4-6% WT, 3-7% YA mutant, 6-9% LLAA mutant, 15% YA/LLAA mutant and 9-10% ∆CT mutant 49K proteins remained at the cell surface. The cell surface expression of some cellular proteins in comparison was only slightly decreased to about 80% of the levels of untreated cells (HLA: ca. 75%, Fas: ca. 86%, EGFR: ca. 80%). Thus, although the internalization of mutated 49K proteins was impaired, 49K staining at the cell surface is still rapidly lost during BFA treatment. One explanation for that finding is that two mechanisms exist that are able to remove WT 49K from the cell surface: Endocytosis and proteolytic cleavage. In case of the mutant 49K proteins, a deficit in endocytosis may result only in a limited accumulation at the cell surface (Fig. 30), since 49K can also be removed from the cell surface by the proteolytic processing.

3.18.3 Increased secretion of E3/49K proteins with mutated cytoplasmic tails

The results from the brefeldin A experiment (Fig. 31) indicated that mutations of the cytoplasmic tail of E3/49K do not prevent proteolytic processing. Therefore, cleavage may occur on the way to and/or at the cell surface. In immunoprecipitation, 49K was detected in cell lines expressing WT and mutant 49K proteins after 2 h labeling (Fig. 32A). Low molecular weight fragments (h) were detected in lysates from cell lines stably expressing mutated E3/49K proteins using antibodies directed against the C-terminus (except for the mutants lacking the cytoplasmic tail which could only be detected with antibodies against the N-terminus). This finding confirmed that the mutant 49K proteins were still proteolytically processed. Notably, the mutations affected the recognition of the cytoplasmic tail of mutated 49K proteins. The YA/LLAA32 clone for example expressed three- to fourfold higher levels of 49K than WT K27S as determined by immunoprecipitation with N-terminal antibody after pulse labeling (Fig. 32B, compare lanes 2 and 15) but is less well precipitated by the antibody against the C-terminus (Fig. 32A, compare lanes 3 and 5). In addition, synthesis rates were measured by quantification of intracellular 49K after brefeldin A treatment by flow cytometry with similar results (data not shown). Interestingly, every mutation changes the migration behavior of the low molecular weight fragments in the polyacryamide gel (Fig. 32A).

To investigate the processing kinetics of WT and mutated 49K, a pulse/chase experiment (30 min pulse, 45 and 180 min chase) with individual clones with similar synthesis rates was performed. No significant differences in the processing kinetics between WT and mutant 49K proteins were observed (Fig. 32C, data not shown). Particularly, the intracellular processing of WT (lane 3-5) and ∆CT (lane 15 –17) 49K proteins was almost identical. The YA/LLAA mutant 49K protein was difficult to precipitate intracellularly (lane 9-11) because of the already described decreased recognition of the mutated cytoplasmic tail with the C-terminal antibody.

97 69 46 30 14 kDa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 mock K9SK27S 30 32 28S2 5 13 29 11S5 5 29 5(αΝ) 29(αΝ)

WT

YA/LLAA

LLAA

YA

∆CT

A