scholarly journals Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2

1997 ◽  
Vol 139 (3) ◽  
pp. 589-599 ◽  
Author(s):  
Sankar Addya ◽  
Hindupur K. Anandatheerthavarada ◽  
Gopa Biswas ◽  
Shripad V. Bhagwat ◽  
Jayati Mullick ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Signal Sequence ◽  
Limited Proteolysis ◽  
Membrane Insertion ◽  
Sequence Motif ◽  
Specific Marker ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Er Membrane

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

scholarly journals A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

1991 ◽  
Vol 112 (5) ◽  
pp. 809-821 ◽  
Author(s):  
R N Thrift ◽  
D W Andrews ◽  
P Walter ◽  
A E Johnson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Secretory Protein ◽  
In Vitro Translation ◽  
Nascent Chain ◽  
Transmembrane Sequence ◽  
Er Membrane

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

scholarly journals Membrane integration of Sec61α: a core component of the endoplasmic reticulum translocation complex

1998 ◽  
Vol 331 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Bruce C. KNIGHT ◽  
Stephen HIGH
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Insertion Site ◽  
Secretory Protein ◽  
Cross Linking ◽  
Central Component ◽  
Membrane Insertion ◽  
Er Membrane

The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61α, Sec61β and Sec61γ, at least two of which (α and β) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61α functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61α is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61α membrane-integration intermediates, we find: (i) newly synthesized Sec61α is adjacent to known components of the ER membrane-insertion site, namely Sec61α, Sec61β and TRAM, and thus the integration of Sec61α appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61α remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61β) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61α requires ER targeting by the signal-recognition particle.

scholarly journals Regulated Targeting of BAX to Mitochondria

1998 ◽  
Vol 143 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Ing Swie Goping ◽  
Atan Gross ◽  
Josée N. Lavoie ◽  
Mai Nguyen ◽  
Ronald Jemmerson ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Transmembrane Domain ◽  
Apoptotic Cell ◽  
Membrane Insertion ◽  
Cell Extracts ◽  
Proapoptotic Protein ◽  
Signal Anchor ◽  
Discrete Domains

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.

scholarly journals Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

2014 ◽  
Vol 13 (4) ◽  
pp. 539-547 ◽  
Author(s):  
VaNae Hamilton ◽  
Ujjal K. Singha ◽  
Joseph T. Smith ◽  
Ebony Weems ◽  
Minu Chaudhuri
Keyword(s):  
Amino Acid ◽  
Alternative Oxidase ◽  
Heterologous Protein ◽  
Amino Acid Residues ◽  
Mitochondrial Targeting ◽  
Content Type ◽  
Targeting Signal ◽  
Internal Signal ◽  
Trypanosome Alternative Oxidase

ABSTRACTRecognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form ofTrypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondriain vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to aT. bruceimitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.

scholarly journals Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel.

1989 ◽  
Vol 109 (5) ◽  
pp. 2033-2043 ◽  
Author(s):  
U C Krieg ◽  
A E Johnson ◽  
P Walter
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
In Vitro Translation ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Glycoprotein ◽  
Messenger Rnas ◽  
Er Membrane

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.

scholarly journals A conserved Guided Entry of Tail-anchored pathway is involved in the trafficking of tail-anchored membrane proteins in Plasmodium falciparum.

2021 ◽  
Author(s):  
Tarkeshwar Kumar ◽  
Satarupa Maitra ◽  
Abdur Rahman ◽  
Souvik Bhattacharjee
Keyword(s):  
Plasmodium Falciparum ◽  
Targeted Delivery ◽  
Transmembrane Domain ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Association Studies ◽  
C Terminus ◽  
N Terminus ◽  
Ta Proteins

Tail-anchored (TA) proteins are defined by the absence of N-terminus signal sequence and the presence of a single transmembrane domain (TMD) proximal to their extreme C-terminus. They play fundamental roles in cellular processes including vesicular trafficking, protein translocation and quality control. Accordingly, TA proteins are post-translationally integrated by the Guided Entry of TA (GET) pathway to the cellular membranes; with their N-terminus oriented towards the cytosol and C-terminus facing the organellar lumen. The TA repertoire and the GET machinery have been extensively characterized in the yeast and mammalian systems, however, they remain elusive in the human malaria parasite Plasmodium falciparum. In this study, we bioinformatically predicted a total of 63 TA proteins in the P. falciparum proteome and revealed the association of their subset with the P. falciparum homolog of Get3 (PfGet3). In addition, our proximity labelling studies either definitively identified or shortlisted the other eligible GET constituents, and our in vitro association studies validated associations between PfGet3 and the corresponding homologs of Get4 and Get2 in P. falciparum. Collectively, this study reveals the presence of proteins with hallmark TA signatures and the involvement of evolutionary conserved GET trafficking pathway for their targeted delivery within the parasite.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3623-3633 ◽  
Author(s):  
J. Jantti ◽  
S. Keranen ◽  
J. Toikkanen ◽  
E. Kuismanen ◽  
C. Ehnholm ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Signal Sequence ◽  
Insertion Site ◽  
Membrane Insertion ◽  
Membrane Anchor

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

scholarly journals Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways

2005 ◽  
Vol 170 (4) ◽  
pp. 595-605 ◽  
Author(s):  
Eric Anderson ◽  
Sandra Maday ◽  
Jeff Sfakianos ◽  
Michael Hull ◽  
Bettina Winckler ◽  
...  
Keyword(s):  
Sequence Motif ◽  
Apical Surface ◽  
Signal Recognition ◽  
Differential Signal ◽  
Spatial Regulation ◽  
A Cell ◽  
Targeting Signal ◽  
Clathrin Adaptor ◽  
Darby Canine Kidney

NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y33RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAM's ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr33. This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.

scholarly journals A cryptic matrix targeting signal of the yeast ADP/ATP carrier normally inserted by the TIM22 complex is recognized by the TIM23 machinery

2004 ◽  
Vol 385 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Maïlys A. S. VERGNOLLE ◽  
Helen SAWNEY ◽  
Tina JUNNE ◽  
Luisita DOLFINI ◽  
Kostas TOKATLIDIS
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Mitochondrial Protein ◽  
Amino Acid Sequences ◽  
Membrane Insertion ◽  
Mitochondrial Targeting ◽  
Terminal Part ◽  
Inner Membrane ◽  
The Matrix ◽  
Targeting Signal

The yeast ADP/ATP carrier (AAC) is a mitochondrial protein that is targeted to the inner membrane via the TIM10 and TIM22 translocase complexes. AAC is devoid of a typical mitochondrial targeting signal and its targeting and insertion are thought to be guided by internal amino acid sequences. Here we show that AAC contains a cryptic matrix targeting signal that can target up to two thirds of the N-terminal part of the protein to the matrix. This event is coordinated by the TIM23 translocase and displays all the features of the matrix-targeting pathway. However, in the context of the whole protein, this signal is ‘masked’ and rendered non-functional as the polypeptide is targeted to the inner membrane via the TIM10 and TIM22 translocases. Our data suggest that after crossing the outer membrane the whole polypeptide chain of AAC is necessary to commit the precursor to the TIM22-mediated inner membrane insertion pathway.

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