Segmental expression and C-terminal labeling of protein ERp44 through protein trans-splicing.

Endoplasmic reticulum resident protein 44 (ERp44) is a member of the protein disulfide isomerase family and functions in oxidative protein folding in the endoplasmic reticulum. A structurally flexible C-terminal tail (C-tail) of ERp44 plays critical roles in dynamically regulating ERp44's function in protein folding quality control. The structure-function dynamics of ERp44's C-tail may be studied further using fluorescence and other techniques, if methods are found to label the C-tail site-specifically with a fluorescent group or segmentally with other desired labels. Here we have developed such methods, employing split inteins capable of protein trans-splicing, and identifying atypical S1 split inteins able to function efficiently at a suitable split site in the ERp44 sequence. One method demonstrated segmental expression of ERp44 for segmental labeling of the C-tail, another method efficiently added a commercially available fluorescent group to the C-terminus of ERp44, and both methods may also be generally useful for studying other proteins.

Segmental Distribution of Myosin Heavy Chain Isoforms Within Single Muscle Fibers.

Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the three EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I⇔IIA⇔IIX⇔IIB, however, in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be because of the myonuclear domain. Anat Rec, 300:1636-1642, 2017. © 2017 Wiley Periodicals, Inc.