The C-terminal domain of the pulmonary surfactant protein C precursor contains signals for intracellular targeting.
We have studied the intracellular transport of the pulmonary surfactant SP-C precursor in vitro and in vivo. In order to monitor the route of the SP-C precursor, we constructed various C-terminally truncated forms of SP-C, which were tagged with a sequence derived from the C-terminus of the human c-myc gene (aa 409-419). Expression of these constructs under the control of the SV40 enhancer and the huMT-II promoter in stably transformed Chinese hamster ovary (CHO) cells revealed that the complete precursor molecule is localized mostly in vesicular structures, probably of lysosomal origin. The truncated precursor lacking the last 22 amino acids at the C-terminus (SP-C/Ctag), however, was restricted to the endoplasmic reticulum as shown by immunofluorescence, using antibodies directed against the tag-sequence, the lysosomal enzyme cathepsin D, the enzyme disulfide isomerase, and the Golgi zone. The intracellular localization was substantiated by subcellular fractionation analysis, suggesting that the last 22 amino acids are necessary for intracellular targeting. Furthermore, Triton X-114 extractions from CHO cells revealed a modification of the SP-C precursor. In vitro translation and pulse-chase experiments in the absence or presence of microsomes showed that the modification occurs post-translationally and in a time-dependent manner. Membrane association studies using an SP-C precursor lacking the mature peptide indicated that the modification is of hydrophobic nature but not a thioester-linked fatty acid.
Keller
A
A
Abteilung für Molekularbiologie, Byk Gulden Pharmazeutika, Konstanz, Germany.
Steinhilber
W
W
Schäfer
K P
KP
Voss
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D011498:Protein Precursors; D011510:Proteolipids; D011663:Pulmonary Surfactants