Htr14 is bound to the plasma membrane and can exist in differently

Htr14 is bound to the plasma membrane and can exist in differently

methylated forms

Immunoblot analysis demonstrated membrane localization for Htr14

A visual inspection of the amino acid sequence of Htr14 identified a hydrophobic region, including residues 28 to 76, that lacks charged amino acids. This stretch could accomodate two transmembrane helices connected by a hairpin loop, as depicted in Fig. 3.14. To determine the cellular localization of Htr14, an antiserum (anti-Htr14D585) was generated against a synthetic

peptide which contains a stretch of amino acids present in the C-terminal region of the protein (D585 to S606). The antiserum is highly specific as demonstrated by the complete absence of reactivity with proteins from the htr14-deletion strain MKK114 (data not shown). Immunoblots of the cytosolic and membrane protein fractions of strains MKK101, S9 and the cheB- and cheR- deletion strains WFS101 and WFS102, respectively, show that the majority of cellular Htr14 pelleted together with the membranes (Fig. 3.10). The small amount of apparently cytosolic Htr14 might result from incomplete pelleting of small membrane fragments. Incomplete pelleting of halobacterial membranes was observed at a growing extent when cells were lysed in buffers of decreasing salt concentration (data not shown). These results suggest that Htr14 is anchored to the membrane, probably via the two predicted N-terminal transmembrane helices. Htr14 can exist in differently methylated forms distinguishable by their

electrophoretic mobilities

To examine the extent of Htr14-methylation, the mobility of Htr14 in SDS-PAGE gels was compared for strains MKK101, S9, WFS101 and WFS102 (Fig. 3.10 A). In strain WFS101 (∆cheB) Htr14 should be fully methylated, because methyl groups can be added by the methyltransferase CheR but not removed by the methylesterase CheB, whereas in strain WFS102 (∆cheR) Htr14 should be completely unmethylated.

The major Htr14 species in the cytosolic as well as in the membrane protein fractions of the investigated strains appear in 3 bands at apparent molecular weights (MW) of approx. 122, 117 and 115 kDa. Of these three bands only the one at 122 kDa is present in strain WFS102. This band is therefore assigned to an unmethylated Htr14 species, consistent with the previous observation that decreasing degrees of methylation of E. coli transducers cause them to run with progressively higher apparent MW (Boyd and Simon, 1980). For strain WFS101 the main band runs at 115 kDa and is attributed to a highly methylated Htr14 species. For strains S9 and MKK101 the strong band at 117 kDa is attributed to a moderately methylated Htr14 species. Above this band another, weaker band at 122 kDa points to the additional presence of an unmethylated Htr14 species in these strains. The faint bands observed in the membrane protein fractions of WFS101 at a MW below 115 kDa and for S9 and MKK101 below 117 kDa might indicate that a minor fraction of the major Htr14 species in these strains is methylated at an additional site. It is unlikely that these bands result from methylation-independent differences in Htr14 mobility, as for strain WFS102 no such band is present below 122 kDa.

The described pattern for the major Htr14 species in the investigated strains is repeated at higher molecular weights. This points to differences between Htr14 species, which cause different mobilities under the used electrophoresis conditions, in addition to the mobility

Figure 3.10 Immunochemical analysis of the cellular localization and the methylation status of Htr14 in different halobacterial strains with anti-Htr14D585 serum.

Cytosolic (cf) and membrane (mf) protein fractions (15 µg protein per lane) were subjected to SDS- PAGE (8% polyacrylamide) followed by immunoblotting as described in Materials and Methods. The relevant strain genotypes concerning the methyltransferase and the methylesterase genes

cheR and cheB, respectively, are indicated in parentheses. Arrows mark the band positions attributed to putatively unmethylated (-), moderately methylated (*) and highly methylated (**) species of Htr14, which are the most prominent (or the only) species in the ∆cheR-, cheBR+_{- and}

∆cheB- strains, respectively.

(A) The majority of cellular Htr14 pellets with the membrane fraction but small amounts are also present in the cytosolic fraction. All depicted lanes are from the same blot with identical exposure times. The pattern of bands at a MW between 115 and 122 kDa is present again at higher MW in the lanes of the membrane fractions. Below the main Htr14 band an additional band is seen with the membrane fraction of the ∆cheB-strain at approx. 113 kDa and with that of cheBR+ _{strain S9 at approx. 115 kDa. (B) Different}

immunoblot, obtained after identical treatment of the protein samples as in A but with 6 M urea additionally present in the separating gel. The methylation-dependent differences in electrophoretic mobility observed in A are abolished. A pattern of three Htr14 species with different mobilities (the lower ones marked by arrows, the very faint upper one by a diamond) is seen for all strains and is also present in A where it is overlaid by the methylation-dependent motility differences.

differences that are due to different extents of Htr14 methylation. Methylation-independent differences in electrophoretic mobility were also reported for E. coli transducers Tsr and Tar (Hazelbauer and Engström, 1981). This might point to an additional, still unspecified type of transducer modification that is conserved among members of the kingdoms of bacteria and archaea. The methylation-dependent mobility differences could be abolished by addition of 6 M urea to the sample buffer and gel, whereas methylation-independent mobility differences persisted (Fig. 3.10 B). A discrepancy between apparent (approx. 122 kDa for unmethylated Htr14) and calculated (65.6 kDa) MW has been noted previously for halobacterial proteins and is thought to be due to their acidic nature (Monstadt and Holldorf, 1991).