SummaryCo-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co-and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co-and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.
SUMMARY
Freeze‐substitution of biological material in pure acetone followed by low‐temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry‐cut sections shows that diffusible cellular thallium ions (Tl+) of Tl+‐loaded muscle are localized at similar protein sites in freeze‐substituted as in frozen‐hydrated preparations. A comparison of X‐ray micro‐analytical data obtained from freeze‐dried cryosections and sections of freeze‐substituted normal (potassium‐containing) muscle shows that K+ ion retention in the freeze‐substituted sample is highly dependent on the freeze‐substitution procedure used; so far, in the best case, about 67% of the cellular K+ is retained after freeze‐substitution in pure acetone and low‐temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze‐substitution and low‐temperature embedding.
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