41594_2022_798_MOESM3

Peer Review Information

Journal

ture

tructural and

olecular

iology

Manuscript Title:

Structurally derived universal mechanism for th

e catalytic cycle of the tail

anchored targeting factor Get3

Corresponding author name(s):

Professor William Clemons

Reviewer Comments & D

ecision

Decision Letter, initial

version:

3rd Feb 2022

Dear Bil,

Thank you again for submitting your manuscript "Structurally d

erived universal mechanism for the

catalytic cycle of the tail

anchored targeting factor Get3". I apologize for the delay in responding, which

resulted from the difficulty in obtaining suitable referee reports. Nevertheless, we now have comments

(below) fr

om the 3 reviewers who evaluated your paper. In light of those reports, we remain interested

in your study and would like to see your response to the comments of the referees, in the form of a

revised manuscript.

I hope you will be pleased to see that all reviewers are positive about the quality and interest of the

study. However, they make detailed suggestions for additional experiments to strengthen some of the

conclusions, and for improving the presentation and

discussion of the findings. Please be sure to

address/respond to all concerns of the referees in full in a point

point response and highlight all

changes in the revised manuscript text file. If you have comments that are intended for editors only,

pleas

e include those in a separate cover letter.

We expect to see your revised manuscript within 6 weeks. If you cannot send it within this time, please

s been accepted for publication at NSMB or published elsewhere.

We are committed to providing a fair and constructive peer

review process. Do not hesitate to contact

us if there are specific requests from the reviewers that you believe are technically imp

ossible or

unlikely to yield a meaningful outcome.

As you already know, we put great emphasis on ensuring that the methods and statistics reported in our

papers are correct and accurate. As such, if there are any changes that should be reported, please

bmit an updated version of the Reporting Summary along with your revision.

Please follow the links below to download these files:

Reporting Summary:

https://www.nature.com/documents/nr

reporting

summary.pdf

Please note that the form is a dynamic ‘smart

pdf’ and must therefore be downloaded and completed in

Adobe Reader.

When submitting the revised version of your manuscript, please pay close attention to our

href="https://www.nature.com/nature

research/editorial

policies/image

integrity">Digital Image

ntegrity Guidelines.</a>

Finally, please ensure that you retain unprocessed data and metadata files after publication, ideally

archiving data in perpetuity, as these may be requested during the peer review and production process

or after publication if an

y issues arise.

If there are additional or modified structures presented in the final revision, please submit the

corresponding PDB validation reports.

SOURCE DATA: we urge authors to provide, in tabular form, the data underlying the graphical

representa

tions used in figures. This is to further increase transparency in data reporting, as detailed in

this editorial (http://www.nature.com/nsmb/journal/v22/n10/full/nsmb.3110.html). Spreadsheets can

be submitted in excel format. Only one (1) file per figure i

s permitted; thus, for multi

paneled figures,

the source data for each panel should be clearly labeled in the Excel file; alternately the data can be

provided as multiple, clearly labeled sheets in an Excel file. When submitting files, the title field shou

indicate which figure the source data pertains to. We encourage our authors to provide source data at

the revision stage, so that they are part of the peer

review process.

Data availability: this journal strongly supports public availability of data. A

ll data used in accepted

papers should be available via a public data repository, or alternatively, as Supplementary Information.

If data can only be shared on request, please explain why in your Data Availability Statement, and also in

the correspondence

with your editor. Please note that for some data types, deposition in a public

repository is mandatory

more information on our data deposition policies and available repositories

can be found below:

https://www.nature.com/nature

research/editorial

polici

es/reporting

standards#availability

data

We require deposition of coordinates (and, in the case of crystal structures, structure factors) into the

Protein Data Bank with the designation of immediate release upon publication (HPUB). Electron

microscopy

derived density maps and coordinate data must be deposited in EMDB and released upon

publication. Deposition and immediate release of NMR chemical shift assignments are highly

encouraged. Deposition of deep sequencing and microarray data is mandatory, and

the datasets must

be released prior to or upon publication. To avoid delays in publication, dataset accession numbers must

be supplied with the final accepted manuscript and appropriate release dates must be indicated at the

galley proof stage.

While we encourage the use of color in preparing figures, please note that this will incur a charge to

partially defray the cost of printing. Information about color charges can be found at

http://www.nature.com/nsmb/authors/submit/index.html#costs

Nature

Structural & Molecular Biology is committed to improving transparency in authorship. As part of

our efforts in this direction, we are now requesting that all authors identified as ‘corresponding author’

on published papers create and link their Open Resea

rcher and Contributor Identifier (ORCID) with their

account on the Manuscript Tracking System (MTS), prior to acceptance. This applies to primary research

papers only. ORCID helps the scientific community achieve unambiguous attribution of all scholarly

ntributions. You can create and link your ORCID from the home page of the MTS by clicking on

‘Modify my Springer Nature account’. For more information please visit please visit <a

href="http://www.springernature.com/orcid">www.springernature.com/orcid</a>.

Please use the link below to submit your revised manuscript and related files:

[

REDACTED

]

<strong>Note:</strong> This URL links to your confidential home page and associ

ated information about

manuscripts you may have submitted, or that you are reviewing for us. If you wish to forward this email

to co

authors, please delete the link to your homepage.

We look forward to seeing the revised manuscript and thank you for the o

pportunity to review your

work.

Kind regards,

Florian

Florian Ullrich, Ph.D.

Associate Editor

Nature Structural & Molecular Biology

ORCID 0000

0002

1153

2040

Referee expertise:

Referee #1: TA pathway

Referee #2: TA pathway, structural biology

Refer

ee #3: TA pathway

Reviewers' Comments:

Reviewer #1:

Remarks to the Author:

In this manuscript, Fry et al. provide a number of Get3 structures and sheds a light on the mechanism of

tail

anchored (TA) protein capture by Get3. Get3 is the conserved ATPase chaperone that post

translationally captures transmembrane domains (TMDs) of T

A proteins with the help of other factors

(Sgt2 and Get4/5). Earlier studies from the author's lab and other labs have solved many different

versions of Get3 including Get3 bound to Get4 or TA protein cargo. Although these studies have

provided important i

nsights into how Get3 captures TMDs of TA proteins, it is still debated how Get3

transforms from an empty state to substrate loaded state, and how this is regulated by ATP. These

questions were challenging to address since available Get3 structures were ob

tained from different

states from different labs. In the current manuscript, the authors aim to address these questions by

using Get3 from Giardia intestinalis. Remarkably, the authors show GiGet3 in nucleotide

free states, ATP

bound states and TA protein

bound states. This series of structures not only confirmed previous

structural studies but also identify novel conformations that take place in GiGet3 with or without TA

protein cargo. For example, in contrast to previous yeast Get3 structures, the authors

find helix5 (H5)

shields the client binding domain (CBD) in GiGet3 and that is displaced when CBD is occupied with the

TMD of TA protein. Overall the manuscript is well organized, and the data are of high quality. The

authors should address and/or discuss

the below concerns to further strengthen the manuscript.

Major comments:

1. The central finding from this study is that the authors discover H5 of GiGet3 plays an important role in

shielding CBD in the apo state as well as shielding the TMD of TA protei

n. Also, interestingly, compare to

other Get3 homologs, GiGet3 has an extended sequence in H5 (126 to 134 amino acids) (Fig S1). This

raises the question of whether the function of H5 shown here is unique to GiGet3 or is conserved to

other Get3 homologs. I

t would be good to delete or mutate H5 in GiGet3 and test if it captures

inefficiently the TMD of TA protein. The authors can do this by co

expressing GiGet3 mutant and TA

protein in E. coli and examining the complex formation as they have done in Fig. S17

2. To show the significance of the author's new findings of H5, I was also wondering H5

mediated

shielding of CBD or TMD might help to capture even suboptimal (less hydrophobic) TA proteins. It would

be good to test a few different TA substrates that va

ry in hydrophobicity from the list that they

identified.

Minor comments:

1. Fig. S11 and Fig. S12 legends ends with helices are numbered as in Fig. ??”

Reviewer #2:

Remarks to the Author:

The manuscript by Fry et al. describes two significant contribu

tions to understanding tail

anchored (TA)

protein insertion by the Guided Entry of TA proteins (GET) pathway. Firstly, the authors identify the GET

factors in the parasite Giardia intestinalis. Secondly, they use X

ray crystallography and single

particle

ryo

EM to determine the conformational landscape of GiGet3. The authors observe that apo Get3 can

adopt two conformations in a single crystal lattice, which both differ from a consensus cryo

structure. They also determine the structure of a GiGet3 ATPas

e inactivating mutant with ATP, which

assumes a slightly different conformation than other Get3 structures bound to ATP analogs. These

conformations inform how intramolecular interactions stabilize mobile elements of Get3 and how Get3

domains move relative

to each other. Perhaps the most exciting finding is the cryo

EM structure of Get3

bound to a TA protein in the post

ATP hydrolysis state which shows how Get3 rearranges to chaperone a

TA protein. The main caveat of the manuscript lies in the limitation to

mostly observational descriptions.

However, a lack of experimental tools for the GiGET system, combined with the structures primarily

revealing conformational differences, may preclude structure

guided experiments. Overall, the analyses

and interpretation

s are thoughtfully and thoroughly considered in the manuscript and generally support

the authors’ claims of identifying the GiGET pathway and establishing the conformational landscape of

Get3 in different nucleotide

bound states as it transitions between b

inding partners.

Main comments:

1. The cryo

EM structure of apo Get3 looks more like the crystal structure of ATP

bound Get3 more than

apo1 and apo2 in Fig. 2. Is it possible that the apo1 and apo2 structures arise from Get3 held by crystal

contacts in co

nformations that are otherwise rarely occupied in biological contexts?

2. Based on the local resolution of the post

hydrolysis Get3 bound to TA protein, additional information

would strengthen the interpretation related to H4/5. Firstly, enforcing C2 symme

try may introduce

artifacts at the axis of symmetry close to the position of H4/5. Can alpha carbons be placed or is this

helical element still clear if symmetry is not enforced? Approximately how long is the substrate TMD and

does this match the Bos1 TMD?

Secondly, it would be useful if the model fitted to map in Fig. 3B was

colored and labeled similarly to Fig. 3D to show the continuity that supports the assignment of the

purple helix as H4/5 instead of other parts of Get3 such as H8.

3. It should be clea

rly noted that GiGet3 is overexpressed with a tag for staining in Fig. 1C. The claimed

cytosolic vs. ER staining in Fig. S5 is also not obvious to me

—

the images all look like disperse spotty

patterns, although this may be due to the resolution of the file.

Since protein dynamics and fine complex

structure is not needed to make this point, is it possible that cytosolic vs. ER staining would be more

clearly distinguished using a lower

resolution confocal such as spinning disk instead of STED?

Reviewer #3:

Remarks to the Author:

Summary of the key results: The manuscript “Structurally derived universal mechanism for the catalytic

cycle of the tail

anchored targeting factor Get3” by Fry and colleagues uncovers components of the

guided

entry of tail

anchored p

roteins (GET) pathway in the pathogenic protist Giardia intestinalis and

provides structural insights into Get3 in different states. The identification of homologs of the pre

targeting complex component Get4, the targeting factor Get3 and the ER

bound GET

receptor

component Get2, support the conservation of the GET targeting pathway to Excavata. Focusing on

GiGet3, the authors use crystallography and cryo

EM to generate a series of structures that provide

insights into how the conformation of this central G

ET pathway component changes upon nucleotide

binding/hydrolysis and client interaction.

Originality and significance, conclusions: The GET pathway is a key ER targeting route for a significant

proportion of membrane proteins, and insights into the conserva

tion of the pathway and mechanistic

details are of broad general interest. Structures of C. thermophilum, S. cerevisiae, S. pombe and human

Get3 in different nucleotide bound/unbound states have been available for some time and already

provide a detailed f

ramework for TA protein targeting by Get3. The key features already identified in

other species are largely conserved to G. intestinialis, but the direct comparison of different states of

the same protein in this study is advantageous. What this manuscript

adds to the understanding of TA

client capture and delivery by Get3 is a more definitive model of how the hydrophobic groove is

occluded upon formation of the close conformation, visualization of the architecture of the post

ATP

hydrolysis state, and an a

lternative view of how shielding of the TA protein during targeting is

accomplished. Beyond the structural analyses, the second aspect of the manuscript focusing on the

conservation of the GET pathway to G. intestinialis is rather preliminary and would ben

efit from further

development.

Data & methodology: The manuscript is well

presented, the majority of the data is of high quality and

the main conclusions are generally well supported. However, the procedures used for indirect

immunofluorescent staining rai

se several questions: The custom

made (?) rat anti

GiGet3 antibody

should be characterized. In the Methods section it states that “an anti

HA tag antibody” from Roche was

used to detect Pdi2 but nowhere is it stated that the G. intestinalis strain used exp

resses an HA

tagged

Pdi2. And in Fig. S5 GiGet3 is HA

tagged and an anti

Pdi2

antibody is mentioned. The catalogue number

for this antibody should be listed. It would be good to create a small Table listing all antibodies and in

which experiment they were

used. All micrographs (Fig. 1C and Fig. S5) lack size bars. If cells were

transfected with tagged constructs for the IF please state in the Legend. The use of statistics is

questionable for the IF experiments since it is not stated how often the experiment

s were repeated and

how many cells were analyzed.

Suggested improvements: 1) The identification of Get3 homologues should be presented more

informatively. The data behind Fig 1B should be made available as a table because the current

visualization does not

allow the individual proteins to be identified. Furthermore, the main

distinguishing features of clade I and II should be explained to rationalize this categorization. The specific

proteins in yeast, humans and G. intestinialis should be marked with arrow

s in the figure.

2) Based on fractionation experiments, it is suggested that GiGet3 primarily localizes to the cytosol.

However, a strong signal for GiGet3 is also detected in the pellet, indicating its association with

membranes. This is in line with the

precipitation of GiGet2 and the microscopy data (Fig. 1C,D) so

should be acknowledged. Replica experiments and quantification would enable the proportion of GiGet3

present at the ER compared to the cytosol to be determined.

3) GiGet2 and GiGet4 were ide

ntified in pulldown assays followed by mass spectrometry and an Sgt2

homolog was found by structure

based homology searching. However, no homologs of Get1 or Get5 are

mentioned. Isolation of complexes via GiGet2 and GiGet4 could reveal these additional hom

ologs as

they would be anticipated to form stable complexes with potential GiGet1 and GiGet5 proteins,

respectively. The identification of GiGet5 is important as, by analogy to other species, this protein would

be expected to mediate hand

over of the TA pr

otein from GiSgt2 to GiGet3. Demonstrating interactions

between Gi

pre

targeting complex components, and also between GiGet5 with GiGet3 would

consolidate that the GET pathway is organized as in other species.

4) The identification of potential clients of

the GiGET pathway is currently based on co

expression of

GiGet3 and GiTA proteins in E. coli, an indirect approach that is prone to artefacts. The conclusion that

GiGet3 targets endogenous TA proteins would be much better supported by showing mislocalizati

on of

such proteins in cells lacking GiGet3, as has been done in other model organisms. Alternatively,

identification of proteins retained with ATPase inactive GiGet3 D53N could provide evidence of TA

protein interacting with GiGet3 in a more endogenous co

ntext.

Minor points:

Page 2, line 51: should read “WRB/CAML in mammals”

Page 6, Legend to Fig. 1C: Pdi2 does not mark the ER membrane but the organelle by staining its lumen

Fig S8: Where the membrane was cut in half should be indicated. Also, for consiste

ncy and to better

visualize GiGet3, the image should be inverted to show black band on a white background.

Author Rebuttal to Initial comments

Reviewers' C

omments:

Reviewer #1:

In this manuscript, Fry et al. provide a number of Get3 structures and sheds a light on the

mechanism of tail

anchored (TA) protein capture by Get3. Get3 is the conserved ATPase

chaperone that post

translationally captures transmembrane domains (TMDs) of T

proteins with the help of other factors (Sgt2 and Get4/5). Earlier studies from the author's

lab and other labs have solved many different versions of Get3 including Get3 bound to

Get4 or TA protein cargo. Although these studies have provided important i

nsights into

how Get3 captures TMDs of TA proteins, it is still debated how Get3 transforms from an

empty state to substrate loaded state, and how this is regulated by ATP. These questions

were challenging to address since available Get3 structures were ob

tained from different

states from different labs. In the current manuscript, the authors aim to address these

questions by using Get3 from Giardia intestinalis. Remarkably, the authors show GiGet3 in

nucleotide

free states,

ATP bound states and TA protein

bound states. This series of structures not only confirmed

previous structural studies but also identify novel conformations that take place in GiGet3

with or without TA protein cargo. For example, in contrast to previous yeast Get3

structures, the authors

find helix5 (H5) shields the client binding domain (CBD) in GiGet3

and that is displaced when CBD is occupied with the TMD of TA protein. Overall the

manuscript is well organized, and the data are of high quality. The authors should address

and/or discuss

the below concerns to further strengthen the manuscript.

Major comments:

1. The central finding from this study is that the authors discover H5 of GiGet3 plays an

important role in shielding CBD in the apo state as well as shielding the TMD of TA protein.

Also, interestingly, compare to other Get3 homologs, GiGet3 has an extended sequence in

H5 (126 to 134 amino acids) (Fig S1).

This raises the question of whether the function of H5

shown here is unique to GiGet3 or is conserved to other Get3 homolog

s. It would be good

to delete or mutate H5 in GiGet3 and test if it captures inefficiently the TMD of TA protein.

The authors can do this by co

expressing GiGet3 mutant and TA protein in E. coli and

examining the complex formation as they have done in Fig.

S17.

The reviewer makes the excellent observation that H5 is longer (9 amino acids) in both the

structure and alignment. Due to manuscript length, we were unable to discuss this in detail.

A notable observation is that the helix length changes across the

crystal forms with the

apoA having the longest H5 while in apoB this extension is disordered due to crystal

packing. This suggests the helix is flexible consistent with its conformational changes and

lack of conservation.

We performed the experiment as requested and have added this as an additional figure

(the new Fig. S23). To be specific, we truncated H5 to be similar to the length of the

metazoan and yeast H5 to directly address the length question. Deleting this helix

ompletely would be incompatible with the post

hydrolysis structure as the H5 helix

additionally contributes to the walls of the hydrophobic groove. In the experiment, the

helix is still capable of forming a complex with the TA client consistent with this d

ifference

not driving the interaction.

Spurred by the suggestion, we were interested in testing the role of H4/5, the loop that

forms the lid in the post

hydrolysis structure, and whether it was required for forming a

stable Get3/TA complex, as one would p

redict from the structure. Deletion of H4/5 resulted

in a loss of complex supporting the model that it was critical for complex stability (Fig S23).

Of note, previous replacement of the H8 “lid” helix (as referred to in the metazoan pre

targeting structure

, Keszei et al.

NSMB

, 2021) with a linker does not affect client binding or

targeting, only client transfer from SGTA to Get3.

2. To show the significance of the author's new findings of H5, I was also wondering H5

mediated shielding of CBD or TMD might he

lp to capture even suboptimal (less

hydrophobic) TA proteins. It would be good to test a few different TA substrates that vary

in hydrophobicity from the list that they identified.

As suggested from the response to the first point, it doesn’t appear that G

iGet3 will have

different properties from the fungal Get3s and will likely have similar binding rules. In the

original draft we had mentioned that we tested a variety of Giardia TA proteins, but only

provided data for two of them. We now have provided data

for all the variants, Fig. S9, and

adjusted our discussion. The putative

TAs selected represent a range of TMD

hydrophobicities (sum of 12.9

31.08 using the TM tendency scale) similar to previous

reports of ER

bound TA proteins in metazoans (12.5

27.3)

(Guna

et al

Science

2018). In the

previous report, zebrafish Get3 was unable to capture and insert TA proteins with

hydrophobicity values less than 22 in an

in vitro

targeting experiment. The list of

Giardia

proteins provided in Table S3 is a putative list complied based on predicted TMDs and

includes TA proteins that are likely mitochondria

bound. Localization in

Giardia

is not as

well characterized as in yeast and metazoans so confidently selecting TAs th

at are ER

bound and Get3 substrates is difficult. Our selection of TAs represents the expected range

of hydrophobicities for ER

bound TA proteins in

Giardia

. Interestingly, the

TA proteins

that bind Get3 and are likely clients (Fig. S9) have high TMD hyd

rophobicities (greater than

the 22 cutoff determined by Guna and colleagues). While an absence of binding in

E. coli

does not mean there is no binding, it is encouraging that the bound clients are the more

hydrophobic TAs. We see no evidence that the longe

r H5 aids in binding less hydrophobic

TA proteins as the strongly bound clients are above the hydrophobic threshold seen in

opisthokonts.

Minor comments:

1. Fig. S11 and Fig. S12 legends ends with helices are numbered as in Fig. ??”

We have fixed this. Thank you for identifying this typo.

Reviewer #2:

The manuscript by Fry et al. describes two significant contributions to understanding tail

anchored (TA) protein insertion by the Guided Entry of TA proteins (GET) pathway. Firstly,

the

authors identify the GET factors in the parasite Giardia intestinalis. Secondly, they use

ray crystallography and single

particle cryo

EM to determine the conformational

landscape of GiGet3. The authors observe that apo Get3 can adopt two conformations i

n a

single crystal lattice, which both differ from a consensus cryo

EM structure. They also

determine the structure of a GiGet3 ATPase inactivating mutant with ATP, which assumes a

slightly different conformation than other Get3 structures bound to ATP ana

logs. These

conformations inform how intramolecular interactions stabilize mobile elements of Get3

and how Get3 domains move relative to each other. Perhaps the most exciting finding is

the cryo

EM structure of Get3 bound to a TA protein in the post

ATP hy

drolysis state which

shows how Get3

rearranges to chaperone a TA protein. The main caveat of the manuscript lies in the

limitation to mostly observational descriptions. However, a lack of experimental tools for

the GiGET system, combined with the structure

s primarily revealing conformational

differences, may preclude structure

guided experiments. Overall, the analyses and

interpretations are thoughtfully and thoroughly considered in the manuscript and generally

support the authors’ claims of identifying the

GiGET pathway and establishing the

conformational landscape of Get3 in different nucleotide

bound states as it transitions

between binding partners.

Main comments:

The cryo

EM structure of apo Get3 looks more like the crystal structure of ATP

bound

Get3 more than apo1 and apo2 in Fig. 2. Is it possible that the apo1 and apo2 structures

arise from Get3 held by crystal contacts in conformations that are otherwise rarel

y occupied

in biological contexts?

Crystal structures represent a likely conformational state seen in solution. The crystal contacts in

our Apo crystal likely capture states that are only transiently occupied in solution. Our

structures represent the range

of conformations apo Get3 can adopt as predicted by single

molecule FRET experiments (Chio

et al

PNAS

, 2017). Based on the number of picked particles

and the final number of particles that resulted in the cryo

EM reconstruction of apo

Get3 we

can estimate that approximately 1.34% of the total number of picked particles used for 3D

classification are in the closed state. This is after some filtering by 2D and 3D classification and

the total number of particles should reflect particles an

d not noise or contaminants. While a

small percentage, the closed apo conformation is likely the most common conformation of apo

Get3. Our work further demonstrates how ATP binding regulates the Get4 interface.

Based on the local resolution of the post

hyd

rolysis Get3 bound to TA protein, additional

information would strengthen the interpretation related to H4/5. Firstly, enforcing C2

symmetry may introduce artifacts at the axis of symmetry close to the position of H4/5. Can

alpha carbons be placed or is th

is helical element still clear if symmetry is not enforced?

Symmetry was imposed at the final step. Without symmetry constraints the reconstruction

refined to 3.87 Å resolution with sharpening as outlined in the Methods section (lines 680

681). New represe

ntative images of the map without symmetry have been added to Fig S20

panels F and G. H4/5 is present in this map and the density reflects a helix (Fig S20G),

although it is important to note that we could not accurately build into this density.

Approximat

ely how long is the substrate TMD and does this match the Bos1 TMD?

The density corresponding to client in the groove is sufficiently long that we estimate it

could accommodate ~22 residues built into a helix, more than sufficient to accommodate

the Bos1 T

MD that is 18 residues long. It is important to note that we are unable to build

accurately into this density. This is likely due to binding of the TMD is based on

hydrophobicity and does not result in a fixed orientation. Reconstructions from single

parti

cle cryo

EM are the result of averaging across thousands of particles. It is likely that the

TMD binds each dimer in slightly different orientations and registries.

Secondly, it would be useful if the model fitted to map in Fig. 3B was colored and labeled

similarly to Fig. 3D to show the continuity that supports the assignment of the purple helix

as H4/5 instead of other parts of Get3 such as H8.

We have edited Fig 3D to make the coloring and labels clearer.

. It should be clearly noted that GiGe

t3 is overexpressed with a tag for staining in Fig. 1C.

There appears to be confusion based on our unclear description. GiGet3 is not

overexpressed in the data shown in Fig. 1C&E where endogenous promotors were used.

In both the immunoprecipitation with the BAP

tag and the STED microscopy the protein

was overexpressed. The methods and figure legend have been adjusted to make this

more ex

plicit.

The claimed cytosolic vs. ER staining in Fig. S5 is also not obvious to me

—

the images all

look like disperse spotty patterns, although this may be due to the resolution of the file.

Since protein dynamics and fine complex structure is not needed to make th

is point, is it

possible that cytosolic vs. ER staining would be more clearly distinguished using a lower

resolution confocal such as spinning disk instead of STED?

The intention of the microscopy analysis was to visualize the colocalization of GiGet3

and

GiGet2. For this reason, both proteins were overexpressed with tags that are

more suitable for STED imaging. Unfortunately, STED analysis enhances membrane

localization over the soluble cytosolic one. The cytosolic localization of GiGet3 is thus

better dem

onstrated by the use of the antibody against the endogenous protein on the

western blot of the cellular fractions.

Reviewer #3:

Summary of the key results: The manuscript “Structurally derived universal mechanism for

the catalytic cycle of the tail

anchore

d targeting factor Get3” by Fry and colleagues uncovers

components of the guided

entry of tail

anchored proteins (GET) pathway in the pathogenic

protist Giardia intestinalis and provides structural insights into Get3 in different states. The

identification

of homologs of the pre

targeting complex component Get4, the targeting

factor Get3 and the ER

bound GET receptor component Get2, support the conservation of

the GET targeting pathway to Excavata. Focusing on GiGet3, the authors use crystallography

and cry

EM to generate a series of structures that provide insights into how the

conformation of this central GET pathway component changes upon nucleotide

binding/hydrolysis and client interaction.

Originality and significance, conclusions: The GET pathway is a

key ER targeting route for a

significant proportion of membrane proteins, and insights into the conservation of the

pathway and mechanistic details are of broad general interest. Structures of C.

thermophilum, S. cerevisiae, S. pombe and human Get3 in dif

ferent nucleotide

bound/unbound states have been available for some time and already provide a detailed

framework for TA protein targeting by Get3. The key features already identified in other

species are largely conserved to G. intestinialis, but the dire

ct comparison of different states

of the same protein in this study is advantageous. What this manuscript adds to the

understanding of TA client capture and delivery by Get3 is a more definitive model of how

the hydrophobic groove is occluded upo

n formation of the close conformation, visualization

of the architecture of the post

ATP hydrolysis state, and an alternative view of how shielding

of the TA protein during targeting is accomplished. Beyond the structural analyses, the

second aspect of the

manuscript focusing on the conservation of the GET pathway to G.

intestinalis is rather preliminary and would benefit from further development.

Data & methodology: The manuscript is well

presented, the majority of the data is of high

quality and the main

conclusions are generally well supported. However, the procedures

used for indirect immunofluorescent staining raise several questions: The custom

made

(?) rat anti

GiGet3 antibody should be characterized.

We have added the following sentence to the Method

s section (Lines 450

454):

“Purified GiGet3 was used as an antigen for in

house production of a polyclonal antibody in

rats. The antibody was validated by western against purified

Get3 and recognized the

same band as a commercial anti

BAP antibody when both were probed against the BAP

tagged

Get3.”

In the Methods section it states that “an anti

HA tag antibody” from Roche was used to

detect Pdi2 but nowhere is it stated that the G

. intestinalis strain used expresses an HA

tagged Pdi2. And in Fig. S5 GiGet3 is HA

tagged and an anti

Pdi2

antibody is mentioned.

The catalogue number for this antibody should be listed. It would be good to create a small

Table listing all antibodies and

in which experiment they were used. All micrographs (Fig. 1C

and Fig. S5) lack size bars. If cells were transfected with tagged constructs for the IF please

state in the Legend. The use of statistics is questionable for the IF experiments since it is not

tated how often the experiments were repeated and how many cells were analyzed.

We modified the Methods sections on the immunofluorescence and STED microscopy to

clarify the cell lines and antibodies used for protein detection. We added scale bars to all

mage series. We have not performed a statistical analysis on the protein localization.

Protein localization was uniform in all confocal and STED images. Representative images

are shown in an updated Figure S6.

Suggested improvements: 1) The identification of Get3 homologues should be presented

more informatively. The data behind Fig 1B should be made available as a table because the

current visualization does not allow the individual proteins to be identified. F

urthermore, the

main distinguishing features of clade I and II should be explained to rationalize this

categorization. The specific proteins in yeast, humans and G. intestinialis should be marked

with arrows in the figure.

We added a table containing the a

ccession numbers and affiliation of the sequences to a

particular clade (Table S1). The categorization is based on the phylogenetic analysis of

all

sequences included into the dataset. The grouping of proteins into two clades is based

on the mutual relationship among the sequences and reflects similarities or differences

present in the initial protein sequence alignment. The presence of two Get3 c

lades has

also been noted in Xing

et al

PNAS

, 2017. Neither analysis identified any motifs specific

to either clade. The differences on the primary sequence level are spread across the

entire sequence which is demonstrated in a new figure (Fig S1).

Based

on fractionation experiments, it is suggested that GiGet3 primarily localizes to

the cytosol. However, a strong signal for GiGet3 is also detected in the pellet, indicating its

association with membranes. This is in line with the co

precipitation of GiGet2

and the

microscopy data (Fig. 1C,D) so should be acknowledged. Replica experiments and

quantification would enable the proportion of GiGet3 present at the ER compared to the

cytosol to be determined.

We thank the reviewer for suggesting the quantification

experiment. Indeed, we agree

that while much of the protein is present in the cytosol, there is a considerable amount

Get3 associated with the ER membrane, which agrees with the protein function. We

have addressed this in the manuscript (Line 130

132

GiGet2 and GiGet4 were identified in pulldown assays followed by mass spectrometry

and an Sgt2 homolog was found by structure

based homology searching. However, no

homologs of Get1 or Get5 are mentioned. Isolation of complexes via GiGet2 and GiGet4

coul

d reveal these additional homologs as they would be anticipated to form stable

complexes with potential GiGet1 and GiGet5 proteins, respectively. The identification of

GiGet5 is important as, by analogy to other species, this protein would be expected to

ediate hand

over of the TA protein from GiSgt2 to GiGet3. Demonstrating interactions

between Gi

pre

targeting complex components, and also between GiGet5 with GiGet3

would consolidate that the GET pathway is organized as in other species.

We agree that a f

ull analysis of the GET pathway components in

Giardia

will be valuable

and an important next step is to identify Get1 and Get5 homologs, if there are any.

While we have found some candidate homologs of Get1 and Get5 in either the genome

analysis or our pro

teomic data, these are not as clear as the other components. Before

making any additional comments on other components we will need to establish

functional studies to identify the role of these proteins. These are complicated

experiments and beyond the sco

pe of this current manuscript. We are excited to

continue these experiments in future studies.

The identification of potential clients of the GiGET pathway is currently based on co

expression of GiGet3 and GiTA proteins in E. coli, an indirect approach tha

t is prone to

artefacts. The conclusion that GiGet3 targets endogenous TA proteins would be much better

supported by showing mislocalization of such proteins in cells lacking GiGet3, as has been

done in other model organisms. Alternatively, ident

ification of proteins retained with ATPase

inactive GiGet3 D53N could provide evidence of TA protein interacting with GiGet3 in a

more endogenous context.

This is indeed one of the crucial experiments. In fact, we have recently established a

CRISPR/Cas9 ge

ne deletion approach in

G. intestinalis

. Unfortunately, we were not able

to obtain a viable

Get3 knockout strain, indicating that the gene might be, in fact,

essential in the protist. We are currently developing an alternative inducible approach.

The strategy of pulling down the D53N mutant would not guarantee positive results as

we do not see cl

ient proteins to be stably bound by

Get3 in the proteomic analysis.

Minor points:

Page 2, line 51: should read “WRB/CAML in mammals”

The human gene for WRB has been renamed by EMBL

EBI as Get1 to be more consistent

with its role in the pathway. We have adopted the new nomenclature.

GENE SYMBOL: GET1

GENE NAME: guided entry of tail

anchored proteins factor 1

SYNONYMS: WRB, CHD5, GET1

Page 6, Legend to Fig. 1C: Pdi2 does not mark the ER membrane but the organelle

by staining its lumen

We have adjusted the legend to reflect this.

Fig S8: Where the membrane was cut in half should be indicated. Also, for consistency and

to better visualize

GiGet3, the image should be inverted to show black band on a white

background.

We have inverted the images and indic

ated where th

e membrane was cut.

Decision Letter, first revision

19th Apr 2022

Dear Bil,

Thank you for submitting your revised manuscript "Structurally derived universal mechanism for the

catalytic cycle of the tail

anchored targeting factor Get3" (NSMB

A45727B). It has now been seen by the

original referees and their comments are below. The r

eviewers find that the paper has improved in

revision, and therefore we'll be happy in principle to publish it in Nature Structural & Molecular Biology,

pending minor revisions to satisfy the referees' final requests regarding the experiment shown in Fig.

S23, and to comply with our editorial and formatting guidelines. Regarding the former, we leave it up to

you if you want to repeat the experiment with the requested controls or if you want to remove the data

from the paper.

We are now performing detailed

checks on your paper and will send you a checklist detailing our

editorial and formatting requirements in about a week. Please do not upload the final materials and

make any revisions until you receive this additional information from us.

To facilitate ou

r work at this stage, we would appreciate if you could send us the main text as a word

file. Please make sure to copy the NSMB account (cc'ed above).

Thank you again for your interest in Nature Structural & Molecular Biology Please do not hesitate to

cont

act me if you have any questions.

Kind regards,

Florian

Florian Ullrich, Ph.D.

Associate Editor

Nature Structural & Molecular Biology

ORCID 0000

0002

1153

2040

Reviewer #1 (Remarks to the Author):

The authors have addressed my concerns. However, Fig.

S23 is not convincing since the absence of the

Get3 signal in dH4/5 could be explained by the low yield of His BRIL Bos1 TMD (bottom) compared to

the trH5 sample. The authors should redo the experiment to obtain a similar recovery of His BRIL Bos1

TMD for

both samples. Alternatively, the authors can remove the data and adjust their conclusion

accordingly.

Reviewer #2 (Remarks to the Author):

The revised manuscript by Fry et al. addresses my original points. My only remaining comment relates to

Fig. S23,

which was added to address a point concerning the importance of the H4/5 loop versus the

length of H5 raised by Reviewer 1. The experiment shown lacks several controls important for

interpretation. It would be useful to see the levels of the Get3 and TA pr

oteins in the input samples and

how recovery of the GiGet3 mutants compares to wildtype. In addition, although the authors pull on the

same TA protein when testing both mutants, the recovery of the TA protein seems to differ by >10

fold

between the two sam

ples. This confounds how much of the difference in GiGet3 recovery can be

attributed to the specific mutants analyzed. From what is shown, I would conclude that the trH5 can

interact with TA protein but would not be confident about conclusions related to t

he H4/5 loop without

additional information. Finally, the Western blot image looks more transparent than expected for the

size of the bands (perhaps during figure formatting?) and appears to be missing from the source data

file. Otherwise, I support public

ation.

Author Rebuttal,

first revision

Reviewer #1 (Remarks to the Author):

The authors have addressed my concerns. However, Fig. S23 is not convincing since the absence of the

Get3 signal in dH4/5 could be explained by the low yield of His BRIL Bos1 TMD (bottom) compared to

the trH5 sample.

The authors should redo the experiment to obtain a similar recovery of His BRIL Bos1

TMD for both samples. Alternatively, the authors can remove the data and adjust their conclusion

accordingly.

We agree with the reviewer and have removed this data and

e corresponding

analysis from our

manuscript.

Reviewer #2 (Remarks to the Author):

The revised manuscript by Fry et al. addresses my original points. My only remaining comment relates to

Fig. S23, which was added to address a point concerning the impor

tance of the H4/5 loop versus the

length of H5 raised by Reviewer 1. The experiment shown lacks several controls important for

interpretation. It would be useful to see the levels of the Get3 and TA proteins in the input samples and

how recovery of the GiG

et3 mutants compares to wildtype. In addition, although the authors pull on the

same TA protein when testing both mutants, the recovery of the TA protein seems to differ by >10

fold

between the two samples. This confounds how much of the difference in GiGe

t3 recovery can be

attributed to the specific mutants analyzed. From what is shown, I would conclude that the trH5 can

interact with TA protein but would not be confident about conclusions related to the H4/5 loop without

additional information. Finally, t

he Western blot image looks more transparent than

expected for the size of the bands (perhaps during figure formatting?) and appears to be missing from

the source data file. Otherwise, I support publication.

As with Reviewer #1, we agree with the reviewer

and have removed this data and

corresponding

analysis

from our manuscript. The source data file was mislabeled FigS20 instead of FigS23

As seen in

the

unprocessed image, there is no manipulation of the western blot as the image was taken directly form

e blot

Final Decision Letter

Dear Dr Fry,

Please find below a copy of the decision letter for your manuscript "Structurally derived universal

mechanism for the catalytic cycle of the tail

anchored targeting factor Get3" [NSMB

A45727C], which

has just been accepted for publication in Nature Structu

ral & Molecular Biology.

The exact publication date will be communicated to the corresponding author. Please note that until

publication, the content of your paper remains under embargo (to determine when the paper can be

discussed with the media, please

consult our embargo policy at

http://www.nature.com/authors/editorial_policies/embargo.html).

To assist our authors in disseminating their research to the broader community, our SharedIt initiative

provides all co

authors with the ability to generate a un

ique shareable link that will allow anyone (with

or without a subscription) to read the published article. Recipients of the link with a subscription will

also be able to download and print the PDF.

As soon as your article is published, you can generate y

our shareable link by entering the DOI of your

article here: <a

href="http://authors.springernature.com/share">http://authors.springernature.com/share<a>.

Corresponding authors will also receive an automated email with the shareable link

If you wish to or

der reprints of your article or have any questions about reprints please send an email to

author

reprints@nature.com.

Please contact the corresponding author directly with any queries you may have related to the content

and publication of your paper.

As we prepare the manuscript for publication, we would like to confirm that your address details are

correct. Could you please click on the link below to verify your profile and correct it as needed? Your

prompt attention to this will help us to avoid dela

ys in publication of your manuscript.

Please verify your address details promptly and correct them as needed by clicking here and following

the link to “Login to My Account/Modify My NRG Profile”:

[

REDACTED

]

You can now use a single sign

on for all your accounts, view the status of all your manuscript

submissions and reviews, access usage statistics for your published articles and download a record of

your refereeing acti

vity for the Nature journals.

Sincerely,

Florian Ullrich, Ph.D.

Associate Editor

Nature Structural & Molecular Biology

ORCID 0000

0002

1153

2040

------------------------------------------------------------------------

Subject: Decision on Nature Struct

ural & Molecular Biology submission NSMB

A45727C

26th May 2022

Dear Bil,

We are now happy to accept your revised paper "Structurally derived universal mechanism for the

catalytic cycle of the tail

anchored targeting factor Get3" for publication as a Art

icle in Nature Structural

& Molecular Biology.

Acceptance is conditional on the manuscript's not being published elsewhere and on there being no

announcement of this work to the newspapers, magazines, radio or television until the publication date

in Natu

re Structural & Molecular Biology.

Over the next few weeks, your paper will be copyedited to ensure that it conforms to Nature Structural

& Molecular Biology style. Once your paper is typeset, you will receive an email with a link to choose the

appropriat

e publishing options for your paper and our Author Services team will be in touch regarding

any additional information that may be required.

After the grant of rights is completed, you will receive a link to your electronic proof via email with a

request

to make any corrections within 48 hours. If, when you receive your proof, you cannot meet this

deadline, please inform us at rjsproduction@springernature.com immediately.

You will not receive your proofs until the publishing agreement has been received th

rough our system.

Due to the importance of these deadlines, we ask that you please let us know now whether you will be

difficult to contact over the next month. If this is the case, we ask you provide us with the contact

information (email, phone and fax)

of someone who will be able to check the proofs on your behalf, and

who will be available to address any last

minute problems.

To assist our authors in disseminating their research to the broader community, our SharedIt initiative

provides all co

authors

with the ability to generate a unique shareable link that will allow anyone (with

or without a subscription) to read the published article. Recipients of the link with a subscription will

also be able to download and print the PDF.

As soon as your articl

e is published, you can generate your shareable link by entering the DOI of your

article here: <a

href="http://authors.springernature.com/share">http://authors.springernature.com/share<a>.

Corresponding authors will also receive an automated email with the

shareable link

Note the policy of the journal on data deposition:

http://www.nature.com/authors/policies/availability.html.

Your paper will be published online soon after we receive proof corrections and will appear in print in

the next available issue.

You can find out your date of online publication by contacting the production

team shortly after sending your proof corrections. Content is published online weekly on Mondays and

Thursdays, and the embargo is set at 16:00 London time (GMT)/11:00 am US Eas

tern time (EST) on the

day of publication. Now is the time to inform your Public Relations or Press Office about your paper, as

they might be interested in promoting its publication. This will allow them time to prepare an accurate

and satisfactory press r

elease. Include your manuscript tracking number (NSMB

A45727C) and our

journal name, which they will need when they contact our press office.

About one week before your paper is published online, we shall be distributing a press release to news

organizati

ons worldwide, which may very well include details of your work. We are happy for your

institution or funding agency to prepare its own press release, but it must mention the embargo date

and Nature Structural & Molecular Biology. If you or your Press Offi

ce have any enquiries in the

meantime, please contact press@nature.com.

You can now use a single sign

on for all your accounts, view the status of all your manuscript

submissions and reviews, access usage statistics for your published articles and downloa

d a record of

your refereeing activity for the Nature journals.

If you have not already done so, we strongly recommend that you upload the step

step protocols

used in this manuscript to the Protocol Exchange. Protocol Exchange is an open online resourc

e that

allows researchers to share their detailed experimental know

how. All uploaded protocols are made

freely available, assigned DOIs for ease of citation and fully searchable through nature.com. Protocols

can be linked to any publications in which they

are used and will be linked to from your article. You can

also establish a dedicated page to collect all your lab Protocols. By uploading your Protocols to Protocol

Exchange, you are enabling researchers to more readily reproduce or adapt the methodology

you use,

as well as increasing the visibility of your protocols and papers. Upload your Protocols at

www.nature.com/protocolexchange/. Further information can be found at

www.nature.com/protocolexchange/about.

An online order form for reprints of your pap

er is available at <a

href="https://www.nature.com/reprints/author

reprints.html">https://www.nature.com/reprints/author

reprints.html</a>. Please let your coauthors

and your institutions' public affairs office know that they are also welcome to order repr

ints by this

method.

Please note that <i>Nature Structural & Molecular Biology</i> is a Transformative Journal (TJ). Authors

may publish their research with us through the traditional subscription access route or make their paper

immediately open access t

hrough payment of an article

processing charge (APC). Authors will not be

required to make a final decision about access to their article until it has been accepted. <a

href="https://www.springernature.com/gp/open

research/transformative

journals"> Find ou

t more

about Transformative Journals</a>

Authors may need to take specific actions to achieve <a