Fig. 1.

Characterizations of retrolinkin (labeled RTLN) as an integral membrane protein. (A) Diagram of mouse retrolinkin protein structure. Amino acid residue numbers denote boundaries for the putative transmembrane (TM) regions in retrolinkin. The cytosolic domain (amino acids 31–460) was used for preparing recombinant domain protein and raising polyclonal antibodies. The fragment of amino acids 173–406 (isolated from yeast two-hybrid screen), containing the site responsible for BPAG1n4's binding, was used for dominant negative assays. (B–D) Expression of retrolinkin. (B) The blot was probed first with antiretrolinkin antibody (lanes 1–4), followed by the purified recombinant domain protein competitions (lanes 1′–4′). After deglycosylation treatment with endo H, the molecular mass of retrolinkin protein was reduced (lane 6). Recom, recombinant protein; br, brain; SC/SN, spinal cord/sciatic nerves; untr, untreated lysates; tr, endo H-treated samples. (C and D) Detections of the protein expression in mouse brain at different developmental stages (C) and in multiple tissues of embryonic day 18 (D) are indicated. Postnatally, retrolinkin could be similarly detected in dorsal and ventral roots of DRGs with NF-L as a neuronal-specific loading control (D, lanes 10 and 11). (E) Endogenous retrolinkin is found primarily in endosomal membrane fractions (lane 4). Spinal cord and sciatic nerve tissue extracts (lane 1) were fractionated by differential centrifugation first into S100 (nuclear component, lane 2) and P100 (membrane fraction, lane 3). The P100 fraction was further fractionated by floatation up through an 8–35–40% sucrose gradient at 150,000 × g for 2 h. Endosomal (lane 4) and heavy (lane 5) membranes were collected from the 8–35% and 35–40% interfaces, respectively. TrkB and Rab5B were used as endosomal vesicular markers, and Lamp2 and Rab7 were used as heavy membrane markers. An aliquot from each step of the fractionations was saved, and the loading was adjusted on equal protein measures. (F) Alkali extraction of retrolinkin. The P100 fraction was treated with 0.1 M Na₂CO3 (pH 11.5) for 30 min on ice and then centrifuged at 100,000 × g for 1 h. Immunoblots were analyzed with antiretrolinkin (Top), APP C terminus (Middle), and EEA1 (Bottom). S, supernatant; P, pellet. (G) The N terminus of retrolinkin resides on the cytoplasmic face of intracellular membranes. Aliquots of total membranes (P100) were treated with 1 μg/ml proteinase K for 20 min at room temperature and then centrifuged at 100,000 × g for 1 h. Immunoblotting was done with antibodies against retrolinkin (Top), APP C terminus (Middle), and N terminus (Bottom).