Abstract. We have used the homobifunctional crosslinking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER . Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles . Two prominent crosslinked products of 45 and 64 kD were detected . The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45-kD product required both SRP and translocation competent microsomal membrane vesicles . In agreement with previous investigators, we suggest that the 64-kD T HE signal sequence of proteins destined for translocatlon across or integration into the rough ER (RER)' contains the information that initiates sequential interactions with cytoplasmic and membrane bound components of the translocation apparatus . The cytoplasmic ribonucleoprotein signal recognition particle (SRP) cotranslationally selects proteins with RER signal sequences by binding to the nascent polypeptide when the signal sequence emerges from the large ribosomal subunit (Walter and Blobel, 19ß1b ;Walter and Blobel, 1982;Walter et al ., 1981) . The signal sequence recognition activity of SRP has been localized to the 54-kD subunit (SRP54) by photo-cross-linking experiments (Krieg et al., 1986;Kurzchalia et al., 1986) . The affinity of the SRP-ribosome complex for the membrane-bound SRP receptor (or docking protein) results in the selective delivery of the ribosome to the membrane surface (Gilmore et al., 19ß2b ; Meyer et al ., 19ß2a ; Walter and Blobel, 198 la), SRP receptor displaces SRP from the signal sequence in a GTPdependent reaction that allows the subsequent membrane insertion of the nascent polypeptide Gilmore and Blobel, 1983) . Although translocation of the polypeptide across the membrane bilayer is a poorly understood process, the preponderance of current experimental evidence suggests that nascent polypeptides undergoing transport are in contact with integral membrane proteins that 1. Abbreviations used in this paper: NEM, N-ethylmaleimide ; RER, rough endoplastnic reticulum; SRP, signal recognition particle .

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Secretion of newly synthesized proteins across the mammalian rough endoplasmic reticulum (translocation) is supported by the membrane proteins Sec61p and TRAM, but may also include accessory factors, depending on the particular translocation substrate. Studies designed to investigate the binding of anti-peptide antibodies to the carboxyl terminus of the alpha-subunit of Sec61 (Sec61palpha) lead us to the isolation of a complex of proteins that occlude the cytosolic face of Sec61palpha in microsomes that have been prepared by standard protocols used to study translocation in vitro [Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93]. This complex was shown by nanospray tandem mass spectrometry to be composed of protein disulfide isomerase (PDI), calcium binding protein 1 (CABP1/P5), 72 kDa endoplasmic reticulum protein (ERp72), and BiP (heat shock protein A5/HSPA5), and has been named TR-PDI for "translocon-resident protein disulfide isomerase complex". This constitutes a novel location for these proteins, which are known to be major constituents of the lumen of the rough endoplasmic reticulum. We have not established the function of TR-PDI at this location, but did observe that the absence of this complex results in a relative loss of correct topology of prion protein insertion across RER membranes, indicating the possibility of a functional role in vivo.

Quantitative fluorescence microscopy is used to compare the binding of the fluorescent, 7‐amino derivative of actinomycin D to a sonication resistant fraction of late‐step mouse spermatids and sperm isolated from three regions of the epididymis. Staining conditions are described that optimize 7‐AMD binding to air‐dried smears of these cells and permit quantitative analyses of 7‐AMD binding to sperm chromatin at the single cell level. These studies show that the step 12–16 spermatid undergoes an 8–10‐fold reduction in 7‐AMD binding as it completes differentiation in the testis and progresses through the epididymis. The first 2–3‐fold reduction in binding, associated with biochemical changes in chromatin structure brought about during spermatid differentiation, occurs gradually and is complete by the time the sperm reaches the caput epididymis. An additional four‐fold reduction is observed as the sperm passes through the corpus. This final alteration, which occurs in a single concerted step, may be reversed by dithiothreitol treatment and appears to be effected through the formation of a final, small group of sperm nuclear protein disulfide crosslinks.

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