Amino terminal sequence of the precursor of ovine β-lactoglobulin

117

The ability of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer into the intravesicular space was investigated by using trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity of microsomal membranes into components that can be separated into two fractions, one soluble and the other membrane bound. The trypsinized membrane fraction has lost its translocation activity. Addition of the trypsin-generated soluble fraction, however, results in reconstitution of translocation activity. These results are compatible with the notion proposed in the signal hypothesis that the translocation activity of the microsomal membrane resides in transmembrane protein(s). We propose that trypsin effects solubilization from the membrane of cytosol-exposed domain(s) involved in recognition of the signal sequence or ribosome or both, leaving behind membrane-integrated domain(s) that provide the environment for the passage of the nascent chain across the membrane. Signal peptidase activity was unaffected by trypsinization of microsomal vesicles consistent with a localization of the active site of this enzyme on the cisternal side of the vesicles.Most of the secretory proteins (1) and several membrane proteins (2-4) so far investigated are synthesized as preproteins containing an amino-terminal extension (signal peptide) when their mRNAs are translated in a cell-free system in the absence of microsomal membranes. The signal peptide portion of the nascent chain has been proposed (5) to contain the necessary information to direct the translating ribosome to form a functional complex with receptor(s) of the rough endoplasmic reticulum membrane and thereby to provide the conditions for a unidirectional translocation of the nascent peptide chain across the membrane into the intracisternal space. Before the nascent chain is completed, the signal peptide is removed by signal peptidase, an endoprotease located on the cisternal side of the membrane (6), except for chicken ovalbumin (7), which is proposed to contain an uncleavable signal sequence (8).Little is known about the recognition-transport machinery in the membrane or the ribosomal components interacting with it. Two membrane proteins, restricted in their location to rough endoplasmic reticulum, have been proposed (9) to function as ribosome receptors, primarily on the basis of their physical association with membrane-bound ribosomes. Furthermore, it has been reported (10) that microsomal membranes can be largely depleted of their translocation activity when extracted with a solution of relatively high ionic strength (500 mM KCl) and that activity can be regenerated when the salt extract is added back to the depleted membranes.We present a way to dissect the translocation activity in the microsomal membrane by limited trypsinization into functionally competent components which were separated by centrifugation. Translocation activity of the membrane can be regenerated by...

mentioning

confidence: 99%

Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes.

Walter¹,

Jackson²,

Marcus³

et al. 1979

117

“…A 20-mer oligomer complementary to PP14 mRNA and corresponding to nucleotides [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] of PP14 cDNA was synthesized and end-labeled with [y-32P]ATP using polynucleotide kinase. The labeled primer (5 ng) was heated with decidual poly(A) RNA (8 gg) for 3 min at 80°C and annealed for 45 min at 61°C.…”

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confidence: 99%

Complete amino acid sequence of human placental protein 14: a progesterone-regulated uterine protein homologous to beta-lactoglobulins.

Julkunen

Seppälä

Jänne

1988

131

Placental protein 14 (PP14), also known as progestagen-dependent endometrial protein and pregnancyassociated endometrial a2-globulin, is synthesized by the human secretory endometrium and decidua. We have isolated from a human decidual cDNA library clones corresponding to PP14 and deduced its entire amino acid sequence. PP14 contains 180 amino acids, 18 of which correspond to a putative signal peptide. The predicted molecular weight of the pre-PP14 is 20,555 and that of the mature protein is 18,787. PP14 is encoded by a 1-kilobase-pair mRNA that is expressed in human secretory endometrium and decidua but not in postmenopausal endometrium, placenta, liver, kidney, and adrenals. The 162-residue-long sequence of PP14 is highly homologous to .8-lactoglobulins, with a 53.4% identity with the amino acid sequence of horse fi-lactoglobulin I. The four cysteinyl residues (positions 66, 106, 119, and 160) responsible for intramolecular disulfide bridges in .-lactoglobulins are all conserved in PP14.

“…Ten of the 13 amino acid residues from positions [8][9][10][11][12][13][14][15][16][17][18][19][20] Biochemistry: Sherwood et (19,(21)(22)(23)(24)(25)(26)(27)(28)(29). From the criteria developed by Segrist and Feldman (30), this region would have a calculated hydrophobicity index of 3.04.…”

Section: Resultsmentioning

confidence: 99%

Primary structure of the NH2-terminal extra piece of the precursor to human placental lactogen.

Sherwood¹,

Burstein²,

Schechter³

1979

The cell-free translation product of human placental lactogen mRNA is a precursor molecule larger than the mature hormone that circulates in plasma. To determine the structure of pre-placental lactogen, the poly(A)-rich RNA fraction of term placenta was isolated and translated in a wheat germ cell-free system. The mRNA programmed the synthesis of a major protein, 3000 daltons larger than placental lactogen, that was specifically precipitated by hormone antibodies. The immunoprecipitated protein was labeled separately with 20 radioactive amino acids and subjected to sequence analysis. The results showed the synthesis of pre-placental lactogen in which an extra piece 25 residues long preceded the NH2 terminus of the mature protein. The structure of the extra piece is as follows:Met-Pro-Gly-Ser-Arg-Thr-Ser-Leu-Leu-Leu-Ala-Phe-Ala-LeuLeu-Cys-Leu-Pro-Trp-Leu-Gln-Glu-Ala-Gl-Ala-.