FIGURE 5.

Water accessibility pattern of unique cysteines in Pen-2 reveals an unexpected topology for Pen-2. A, amino acid (AA) sequence of murine Pen-2 is shown. The amino acids that were mutated to cysteine are indicated. Putative hydrophobic domain 1 and 2 of Pen-2 are displayed as α-helices according to the predictions of TOPPRED. An overview of the results of the water accessibility assays is shown in A. Dark and light gray circles represent cysteines with high or low reactivity towards the biotinylated reagents, respectively. Light ringed white circles represent cysteines that do not react with the biotinylated reagents. B, intact cells were exposed to the membrane-permeable sulfhydryl-specific reagent EZ-linked biotin-HPDP (upper panels) or the membrane-impermeable sulfhydryl-specific reagent MTSEA-biotin (lower panels). Biotinylated proteins were precipitated by neutravidin beads in the presence of Triton X-100. Input and bound fractions were separated by SDS-PAGE (4–12% BisTris) and Western blotting, and Pen-2 was detected using the B126 antibody recognizing the N terminus. Wild type Pen-2 (WT) with its lumenal cysteine at the N terminus was used as a positive control; cysteine-less Pen-2 (CL) was a negative control. C, as a control for the reliability of the MTSEA reagent, intact cells were treated with the membrane-impermeable TS-biotin-XX in the same way as in B. The Pen-2 cysteine mutant E49C shows reactivity to the impermeable TS-XX-biotin, confirming the results of the MTSEA-biotin reagent. D, pretreatment with the bulky and charged MTSES-reagent results in a decreased MTSEA-biotin labeling of the cysteine at the C terminus of Pen-2 (W85C). In contrast, the endogenous cysteine at the N terminus of Pen-2 (Cys¹⁵) does not display labeling differences with or without pretreatment, indicating that this cysteine is present in a restricted environment. Pretreatment with MTSES did not change the labeling with MTSEA-biotin for the cysteine in the N-terminal part of hydrophobic domain 1 (F25C) either. E, model of the mode of action of the different sulfhydryl-specific reagents used in this research. The different positions (numbers 1–5) of cysteines in a hypothetical membrane spanning protein and their labeling (+ or −) by the sulfhydryl-specific reagents used in this study are indicated.